恒温扩增型表面等离子体共振生物传感器构建与结核杆菌及其多重耐药突变位点检测研究
|
摘要
背景
近年来，由于人口数量及流动性增加、诊治延误和抗结核药物的不规范使用，以及艾滋病等免疫缺陷病的流行，使得结核耐药日趋严重，耐多药结核(MDRTB)甚至广泛耐药结核(XDRTB)严重蔓延，严重危害社会公共卫生安全。据WHO统计数据表明，在全球22个结核及耐药结核高负担国家中中国位列第二，耐药结核感染率全球第一。对于耐药结核患者，早发现、早治疗是取得成功治疗的关键。在制定治疗方案时，最好根据药敏试验结果进行个体化治疗。对结核分枝杆菌及其耐药检测的传统方法，主要依靠痰涂片、培养、药敏、PCR技术等多项试验综合分析，方法繁琐、需时长、阳性率低、特异性差，提供的耐药信息量少；而目前基于耐药基因检测的分子生物学技术，如直接测序法、PCR-SSCP、微阵列基因芯片法等，价格昂贵，操作要求高。因此，建立新型检测结核菌耐药基因型的方法，用于快速检测耐药结核菌，对结核病早期治疗和控制耐药菌的传播具有重要意义。
表面等离子体共振(surface plasmon resonance，SPR)传感器是近年来迅速发展起来的新型光学传感器，其原理在于当光在棱镜与金属膜表面上发生全反射现象时，其对应的入射角即SPR角与金属表面结合的分子质量成正比。它具有实时、无需标记、在线分析等优势，可通过换能器，将传感器芯片表面配体与分析物作用的生物信号转换为可检测的光电物理信号，实现对样本中靶分子的检测。在传感器相关检测中以PCR为代表的核酸扩增技术常被用于提高检测限。但由于复杂的热循环过程带来的非特异性扩增、与芯片表面扩增的不兼容性以及增加的检测成本，仍然是需要考虑的问题。RCA是一种恒温的DNA扩增过程，可于短时间内在固相载体表面扩增出上千倍互补与环状模板的串联重复序列。与传统PCR相比，RCA具有无需循环控温、线性扩增、扩增产物易于固定等优势，近年来成为一种新颖的信号扩增工具用于DNA等痕量物质的超敏检测。恒温连接酶的特异性使该方法对单碱基突变有较高的敏感度和分辨率，具有链置换活性的多聚酶使该扩增反应能在恒温条件下进行，且靶序列仅作为生成环状模板的支架，避免了可能的扩增污染和潜在的生物危害。
本研究结合课题组前期的研究基础，将芯片表面锚定RCA与SPR生物传感技术相整合，建立了一种新型的恒温扩增型SPR生物传感器阵列，利用自行设计的标签型锁式探针(Padlock Probe, PLP)，可实现多重点突变的恒温、高通量、无标记检测。并将其运用于结核杆菌耐药相关基因突变位点的多重检测，可直接对结核杆菌核酸及其多重耐药基因的5种常见突变位点进行恒温快速检测，为结核分枝杆菌及耐药基因快速鉴定和流行病学调查等提供坚实的实验基础。
方法
1．利用磁珠颗粒模拟固相反应体系，根据生物配体的共价偶联将氨基修饰的寡核苷酸探针(捕获探针)固定于羧基修饰的磁珠表面构成固相反应生物敏感膜，探讨固相RCA反应的具体方式和最优反应体系。利用SRBR-Green II对液相RCA扩增体系进行荧光定量观察，以确定液相扩增反应的最佳体系。
2.在前期研究基础上，对SPR生物传感器检测平台的硬件设施和软件设施进行改进，增加检测通道，改进温控和流速控制系统及分析物流通方式。对传感器芯片自组装相关方法进行改进并制备生物敏感膜。
3. NCBI数据库查找针对四种临床常用一线抗结核药物的5种耐药基因主要突变位点。利于Primer Premier5.0设计包含标签序列、通用序列、两端识别序列的锁式探针(PLP)，并对形成的环状探针进行二级结构预测和分析。利于Array Designer4.0软件，设计包含标签序列探针，使各探针间保持较好的一致性和特异性。探讨杂交时间、杂交温度、扩增时间等各项反应条件对SPR信号值的影响，确定芯片表面RCA反应的最佳条件。
4.将纳米金信号放大与固相表面锚定RCA反应相结合，通过纳米金颗粒修饰的通用序列探针与固相表面RCA反应产物的大量串联重复序列杂交，从而产生具有强大信号放大能力的联级信号放大效果。优化纳米金信号放大系统的反应条件，并以人工合成的模式DNA分子检测该反应系统的灵敏度和特异性。
5.根据GenBank找到的结核分枝杆菌16s-23s rRNA基因ITS序列，设计包含硫代磷酸化修饰的酶切位点的锁式探针，建立基于靶引物的液相RCA反应鉴定结核与非结核分枝杆菌SPR生物传感器检测方法，并实现液相RCA反应的无标记实时动态监测。
6．以34例临床标本为检测对象，针对四种一线抗结核药物的五个突变位点进行MDRTB临床标本的SPR生物传感器检测，并与测序结果对照；对建立的恒温型SPR生物传感器微阵列检测方法进行方法学比较和评估，并确定临床样本检测步骤。
结果
1.完成以磁珠为载体的固相RCA体系构建，并确定以液相连接后固相载体表面扩增的方式进行反应。固相RCA的最佳反应体系为0.5U/μL Phi29DNA polymerase，0.2μg/μL BSA，5%DMSO，1mM dNTPs。连接产物及RCA产物电泳结果显示，仅在突变型靶序列存在条件下生成的环状模板可引发固相表面RCA反应，反之则无产物生成。
2.成功构建新型恒温固相扩增型SPR生物传感器检测仪，传感器芯片为20mm x28.6mm镀金膜棱镜，检测流池为8通道串联流通池。系统温度控制范围为25℃-60℃，温控精度±0.1℃；流速范围为5-2000μL/min，流速控制精度为±1μL/min；检测池在反应中能保持稳定负压，以保证系统反应的稳定性；采用巯基末端修饰共价结合法将还原型捕获探针自组装于芯片金膜表面，探针浓度为1μM。
3.利用自行设计的锁式探针可以完全分辨靶序列单碱基突变，RCA可将SPR信号值(突变位点信号)放大10.0倍，突变识别率为5000：1(野生型与突变型靶序列的浓度比)。连接产物、RCA产物、以及RCA产物HhaI酶切电泳条带位置准确，明亮清晰。自行设计的捕获探针特异性好、各探针之间具有相同的长度和相近的Tm值(<1℃)，可使各探针在相同条件下杂交，适于传感器微阵列系统的多通道并行检测。检测最适液相靶序列杂交温度为45℃，芯片表面杂交温度为37℃，RCA扩增时间为30min。
4.针对5种临床常见结核杆菌耐药主要突变位点：异烟肼(INH)：KatG315(AGC→ACC), inhA-15(ACG→ATG)；利福平(PFP)：rpoB526(CAC→TAC)；乙胺丁醇(EMB)：embB306(ATG→GTG)；链霉素(SM)：rpsL43(AAG→AGG)，设计包含标签型锁式探针及相应捕获探针的探针组，并实现了芯片表面各突变位点的多重扩增检测，同一突变位点对突变型和野生型靶序列的SPR信号值差异明显(p <0.01)，各突变位点间交叉反应结果显示SPR信号值差异明显(p <0.01)。
5．建立的纳米金信号放大系统可有效提高检测的灵敏度，直径15nm的纳米金颗粒在pH7.5和8%工作浓度下可将SPR信号值(RCA放大后)放大2.1倍，与阴性和空白对照信号值比较有显著性差异(p <0.01)。对合成靶序列的检测检测限为5X10~(-12)M (59.1±7.1mo)，且在10-12M至10~(-8)M浓度范围内成线性相关(y=615.29x+1323.8, R2=0.9846)。
6.建立的液相target-primed RCA SPR生物传感器检测系统，可通过两步法在4h内完成对结核分枝杆菌群复合群(MTBC)和鸟型分支杆菌群复合群(MAC)的恒温(37℃)快速检测。SPR方法对临床标本的检测结果与菌种鉴定结果一致。最低检测限分别为MTBC：4.2X10~4CFU/mL (0.005ng/μL)，MAC：3.7x10~4CFU/mL (0.002ng/μL)；经特殊设计的锁式探针可使液相RCA反应与限制性内切酶反应同时进行，实现无标记的SPR实时动态监测RCA反应。
7.采用试剂盒法和加热碱裂解法对24例临床分离株标本和10例临床分泌物样本提取基因组DNA结果显示，碱裂解法操作简便，对临床分泌物标本和临床分离株均有较好的提取效果，可作为样本的前处理方法。SPR传感器方法检测34例临床标本，检测限为8.2pg/μL (65.7±8.5mo)临床标本基因组DNA，临床灵敏度和特异性与测序方法比较分别katG315：92%和80%；inhA-15：100%和100%；rpoB526：94.4%和100%；embB306：95%和100%；rpsL43：100%和100%。经Kappa检验两种方法检测结果具有较好的一致性性差异(p <0.05, Kappa>0.75)。
结论
1.新型恒温扩增型SPR生物传感器，改变了普通高灵敏性传感器检测对PCR方法的依赖。其稳定密闭的检测系统和简单低耗能的操作，有利于传感器的小型化和便携性。
2.自行设计的标签型锁式探针，不仅可在高背景干扰下识别突变靶序列，还可实现混合靶序列的多重连接反应。其包含的标签序列，可实现芯片表面的多重固相扩增反应，且反应条件一致，交叉反应小，增加了传感器微阵列的检测通量。
3.纳米金信号放大系统可显著增加SPR芯片表面的等离子共振强度，有效提高传感器的检测灵敏度，为SPR生物传感器的痕量物质检测提供了一种可靠的信号放大方法，在核酸非扩增快速检测及生物传感器领域有较大的应用潜力。
4.基于靶引物的液相RCA型SPR生物传感器检测系统，在保证特异性与敏感性的基础上，可快速恒温地实现对液相扩增反应的无标记实时动态监测，为核酸扩增检测提供了一条新思路。
5.构建的新型SPR生物传感器检测系统，可在4小时内同时检测五种临床常见MDRTB耐药突变位点，检测方法具有灵敏、特异、操作简单、快速的特点。与传统基因芯片相比，极大降低了技术、设备要求和检测成本。为临床结核杆菌耐药的恒温快速检测乃至其他基因检测提供一种新方法新思路，有望弥补现有常规检测方法存在的不足，满足临床快速诊断的需求，具有广阔的应用前景。
Background
Due to the increase of population number and mobility, non-standard drug-use, as wellas the HIV/AIDS pandemic, a steady drug-resistant increase of M.tuberculosis hasthreatened tuberculosis (TB) control world-widely, despite advancements in the diagnosisand treatment of TB over the years. The multidrug-resistant strains were defined as beingresistant to at least isoniazid and rifampin. The emergence and spread of multidrug-resistantMycobacterium tuberculosis (MDRTB) even extensively drug resistant tuberculosis(XDRTB) posed an upward trend and represented an increasing public health problem.Conventional culture-based bacterial identification method or sequence analysis canprovide definitive results, however, it is time-consuming, laborious and expensive.Therefore, the development of simple, sensitive and low-cost method is important for therapid detection of drug-resistance. It is a timely and effective way to diagnose and manageTB patients, especially MDRTB infected.
Because of high sensitivity of optical transducer, convenient operation and real-timemonitoring, surface plasmon resonance (SPR) biosensors have been adopted in numerousstudies on variety of bimolecular interaction including DNA, RNA, protein and bacteriumbased on different recognition elements. In a SPR biosensor assay, the change of therefractive index on the gold film correlates with the amount of analyte bound to the film. Inmost of SPR-based DNA detection with high sensitivity, nucleic acid amplification isadopted to amplify analyte mass, especially polymerase chain reaction (PCR). However, thenonspecific amplification from complicated thermal circling step, non-compatibility withon-chip amplification and increase of detection cost are still problems to be considered.
Focusing on the development of isothermal amplification methods, RCA has shown itsattraction due to the specificity and multiplexing besides simplicity. The L-RCA by ligase relies on base pairing principle which requires perfect complementarity on the ligation nick.It not only forbids the mismatch but also has a low occurrence of false positive results whencompared to PCR. When used as a signal amplification method, this property enabled highmultiplexing without interference, direct amplification on solid phase and precise productlocalization. Moreover, RCA amplifies the circular PLP only, without accumulation oftarget templates over time, which minimizes the risk of contamination and the potentialbiohazard.
In this study, we developed a SPR DNA biosensor method combined withsurface-anchored RCA to isothermally detect multiple point mutations associated withdrug-resistance in MDRTB. The Au nanoparticle signal amplification was adopted tofurther improve the sensitivity. The sensitivity and specificity of the SPR detection systemwere evaluated and the application was tested in real clinical samples. Besides, we alsodeveloped a SPR real-time detection method by target-primed RCA to discriminate TBbacilli from nontuberculous mycobacteria. This study lays the groundwork for applying theSPR biosensor for isothermal and rapid detection of resisitence associated mutations inM.tuberculosis.
Methods
1.The solid-phase RCA mode was contrasted by using magnetic beads. Theamino-modified oligonucleotide probe (capture probe) was immobilized on the surface ofthe carboxyl-modified magnetic beads through covalent coupling principle to form sensingmembrane. The optimum detection procedure and reaction condition were investigated. Theliquid phase RCA reaction condition was also investigated by the fluorescence quantitativemethod with SRBR Green II dye.
2.Based on the previous study, the SPR biosensor platform was constructed withimprovements to increase the detection channel, improve temperature control, flow controland analyte circulating ways. The control software UMPHO-A600was designed tointerface with the SPR detector. The stability and reproducibility were evaluated. Theself-assembly of the sensor chip was improved to prepare microarrays.
3.The sequences of five clinically meaningful mutations in MDRTB were searched forin the NCBI database. The padlock probe (PLP) containing a specific tag-sequence, ageneral sequence, and a target-specific region on each detection arm, was designed with Primer Premier5.0software. The capture probe containing tag-sequence with consistencyand specificity was designed by Array Designer4.0. The second structure prediction of thecircular PLP was also analyzed.
4.Combination of Au nanoparticle signal amplification with the surface-anchored RCAresulted in cascade signal amplification on the chip surface. The experimental conditionsfor the detection system were also optimized. The synthetic oligonucleotides were used toevaluate the sensitivity and specificity of the detection system.
5.According to the sequence of ITS in16s-23s rRNA gene found in GenBank, PLPwas designed to contain the restriction cutting site. The target-primed RCA was establishedto discriminate M.tuberculosis from NTM with the SPR biosensor. The dynamic real-timemonitoring of liquid-phase RCA with label-free manner was achieved.
6.The detection system was demonstrated by targeting five resistance associatedmutations in MDRTB from34cased clinical samples. The sequencing was made asreference. The sensitivity and specificity of the developed SPR biosensor system formultiplex mutation detections of clinical samples were tested.
Results
1.The solid-phase RCA system was achieved on the carrier of magnetic beads and theamplification way was determined as ligation in solution combined with surfaceamplification. The optimum solid-phase RCA reaction condition was determined as0.5U/μL Phi29DNA polymerase，0.2μg/μL BSA，5%DMSO，1mM dNTPs. Theelectrophoresis verification showed that the circular PLP could be generated only in thepresence of mutant target to cause the following surface RCA reaction, whereas no reactionhappened.
2.The sensor chip was the gold film prim with the size of20mm x28.6mm. Thedetection well was eight-channel tandem. The temperature was controlled with an accuracyof±0.1℃at25~60℃. The flow rate could be changed from5μL/min to2000μL/min withan accuracy of1μL/min. the detection well was in negative pressure to form a sealcondition to maintain the stability of the reaction during reaction. There was scarcelyinterference between channels to achieve the array detection. The thiol group modifiedcapture probes were self-assembled on the chip surface through the covalent binding withthe concentration of1μM.
3.The designed PLP can distinguish the target with single-base mismatch. RCA canamplify the SPR signal for almost10-fold. The positive mutation detection is achieved witha wild-type to mutant ratio of5000:1. The electrophoresis bands of ligation products, RCAproducts and restriction enzyme products of RCA were accurate and clear. The designedcapture probes with the similar length and Tm values (<1℃) can react under the samecondition, but also owned the high specificity. The optimum hybridization temperature inligation was45℃, while on the chip surface was37℃. The RCA duration time was30min.
4.The probe sets were designed to target five common clinical resistance associatedmutation site, including isoniazid (INH): KatG315(AGC→ACC), inhA-15(ACG→ATG); rifampicin (PFP): rpoB526(CAC→TAC); ethambutol (EMB): embB306(ATG→GTG); streptomycin (SM): rpsL43(AAG→AGG). The multiplex amplification on chipsurface was achieved. The SPR signal difference between mutant and wild-type target wassignificant (p <0.01). The cross reaction result showed that the SPR signal differencebetween each mutation site was significant (p <0.01).
5.The Au nanoparticle signal amplification system could effectively improve thesensitivity for2.1-fold, with15nm diameter, pH7.5and8%of concentration, whichshowed significant difference (p <0.01) compared to the negative control and blank control.The detection limit was5x10-12M synthetic oligonucleotides, and a linear correlation (y=615.29x+1323.8, R2=0.9846) was found within the concentration range of10-12M to10-8M.
6.The developed target-primed RCA SPR biosensor system to isothermally (37℃)and rapidly (within4h) detect MTBC and MAC could be performed by only two steps. Theresults of clinical samples from SPR method and bacteria identification were consistence.The detection limit for MTBC was4.2X104CFU/mL (0.005ng/μL), and MAC was3.7x104CFU/mL (0.002ng/μL), respectively. The specially designed PLP could make theliquid-phase RCA and digestion reaction perform simultaneously, which achieve thedynamic real-time monitoring of RCA by SPR biosensor.
7.The heated alkali lysis method was more simple, cost-effective and sensitive toextract genomic DNA in clinical samples. Compared to the sequencing method, thesensitivity and specificity of the SPR detection were92%and80%, respectively, for katG 315,100%and100%, respectively, for inhA-15,94.4%and100%, respectively, for rpoB526,95%and100%, respectively, for embB306,100%and100%, respectively, for rpsL43(Table2). According to the Kappa test,(p <0.05, Kappa>0.75) there exists significantconsistency between the two methods.
Conclusions
1.The developed isothermal amplification based SPR biosensor changed the detectionmethod of PCR-dependent traditional biosensors. The sealed and stable detection systemwith convenience and low power consumption was favor of miniaturization and portabilityapplication.
2.The designed PLP had excellent capability of detecting the low-abundance mutationin the large background of wild targets, but also achieved the multiplex ligation with mixedtargets. The tag-sequence achieved the multiplex surface-anchored RCA with same reactioncondition and no interference, improved the multiplexing capabilities of the biosensor arrayand simplified the operation.
3.Au nanoparticle signal amplification system could significantly increase the plasmonoscillations with a resonant frequency, thus improve the signal/noise ratio and thesensitivity. This system provided a reliable signal amplification method to detect traceanalyte, and hold great promise in scientific and clinical diagnostic application.
4.The target-primed RCA SPR biosensor system achieved dynamically real-timemonitor the liquid amplification with label-free manners, which provided a rapid andisothermal detection method with specificity and sensitivity.
5. Compared with the sequencing method, the developed SPR biosensor detectionsystem has good sensitivity and specificity in the detection of clinical samples, whichindicated that the novel SPR biosensor based on isothermal amplification would be apotential clinical diagnostic method for rapid detection of multiple resistance associatedmutation or other mutations associated disease in clinical samples
近年来，由于人口数量及流动性增加、诊治延误和抗结核药物的不规范使用，以及艾滋病等免疫缺陷病的流行，使得结核耐药日趋严重，耐多药结核(MDRTB)甚至广泛耐药结核(XDRTB)严重蔓延，严重危害社会公共卫生安全。据WHO统计数据表明，在全球22个结核及耐药结核高负担国家中中国位列第二，耐药结核感染率全球第一。对于耐药结核患者，早发现、早治疗是取得成功治疗的关键。在制定治疗方案时，最好根据药敏试验结果进行个体化治疗。对结核分枝杆菌及其耐药检测的传统方法，主要依靠痰涂片、培养、药敏、PCR技术等多项试验综合分析，方法繁琐、需时长、阳性率低、特异性差，提供的耐药信息量少；而目前基于耐药基因检测的分子生物学技术，如直接测序法、PCR-SSCP、微阵列基因芯片法等，价格昂贵，操作要求高。因此，建立新型检测结核菌耐药基因型的方法，用于快速检测耐药结核菌，对结核病早期治疗和控制耐药菌的传播具有重要意义。
表面等离子体共振(surface plasmon resonance，SPR)传感器是近年来迅速发展起来的新型光学传感器，其原理在于当光在棱镜与金属膜表面上发生全反射现象时，其对应的入射角即SPR角与金属表面结合的分子质量成正比。它具有实时、无需标记、在线分析等优势，可通过换能器，将传感器芯片表面配体与分析物作用的生物信号转换为可检测的光电物理信号，实现对样本中靶分子的检测。在传感器相关检测中以PCR为代表的核酸扩增技术常被用于提高检测限。但由于复杂的热循环过程带来的非特异性扩增、与芯片表面扩增的不兼容性以及增加的检测成本，仍然是需要考虑的问题。RCA是一种恒温的DNA扩增过程，可于短时间内在固相载体表面扩增出上千倍互补与环状模板的串联重复序列。与传统PCR相比，RCA具有无需循环控温、线性扩增、扩增产物易于固定等优势，近年来成为一种新颖的信号扩增工具用于DNA等痕量物质的超敏检测。恒温连接酶的特异性使该方法对单碱基突变有较高的敏感度和分辨率，具有链置换活性的多聚酶使该扩增反应能在恒温条件下进行，且靶序列仅作为生成环状模板的支架，避免了可能的扩增污染和潜在的生物危害。
本研究结合课题组前期的研究基础，将芯片表面锚定RCA与SPR生物传感技术相整合，建立了一种新型的恒温扩增型SPR生物传感器阵列，利用自行设计的标签型锁式探针(Padlock Probe, PLP)，可实现多重点突变的恒温、高通量、无标记检测。并将其运用于结核杆菌耐药相关基因突变位点的多重检测，可直接对结核杆菌核酸及其多重耐药基因的5种常见突变位点进行恒温快速检测，为结核分枝杆菌及耐药基因快速鉴定和流行病学调查等提供坚实的实验基础。
方法
1．利用磁珠颗粒模拟固相反应体系，根据生物配体的共价偶联将氨基修饰的寡核苷酸探针(捕获探针)固定于羧基修饰的磁珠表面构成固相反应生物敏感膜，探讨固相RCA反应的具体方式和最优反应体系。利用SRBR-Green II对液相RCA扩增体系进行荧光定量观察，以确定液相扩增反应的最佳体系。
2.在前期研究基础上，对SPR生物传感器检测平台的硬件设施和软件设施进行改进，增加检测通道，改进温控和流速控制系统及分析物流通方式。对传感器芯片自组装相关方法进行改进并制备生物敏感膜。
3. NCBI数据库查找针对四种临床常用一线抗结核药物的5种耐药基因主要突变位点。利于Primer Premier5.0设计包含标签序列、通用序列、两端识别序列的锁式探针(PLP)，并对形成的环状探针进行二级结构预测和分析。利于Array Designer4.0软件，设计包含标签序列探针，使各探针间保持较好的一致性和特异性。探讨杂交时间、杂交温度、扩增时间等各项反应条件对SPR信号值的影响，确定芯片表面RCA反应的最佳条件。
4.将纳米金信号放大与固相表面锚定RCA反应相结合，通过纳米金颗粒修饰的通用序列探针与固相表面RCA反应产物的大量串联重复序列杂交，从而产生具有强大信号放大能力的联级信号放大效果。优化纳米金信号放大系统的反应条件，并以人工合成的模式DNA分子检测该反应系统的灵敏度和特异性。
5.根据GenBank找到的结核分枝杆菌16s-23s rRNA基因ITS序列，设计包含硫代磷酸化修饰的酶切位点的锁式探针，建立基于靶引物的液相RCA反应鉴定结核与非结核分枝杆菌SPR生物传感器检测方法，并实现液相RCA反应的无标记实时动态监测。
6．以34例临床标本为检测对象，针对四种一线抗结核药物的五个突变位点进行MDRTB临床标本的SPR生物传感器检测，并与测序结果对照；对建立的恒温型SPR生物传感器微阵列检测方法进行方法学比较和评估，并确定临床样本检测步骤。
结果
1.完成以磁珠为载体的固相RCA体系构建，并确定以液相连接后固相载体表面扩增的方式进行反应。固相RCA的最佳反应体系为0.5U/μL Phi29DNA polymerase，0.2μg/μL BSA，5%DMSO，1mM dNTPs。连接产物及RCA产物电泳结果显示，仅在突变型靶序列存在条件下生成的环状模板可引发固相表面RCA反应，反之则无产物生成。
2.成功构建新型恒温固相扩增型SPR生物传感器检测仪，传感器芯片为20mm x28.6mm镀金膜棱镜，检测流池为8通道串联流通池。系统温度控制范围为25℃-60℃，温控精度±0.1℃；流速范围为5-2000μL/min，流速控制精度为±1μL/min；检测池在反应中能保持稳定负压，以保证系统反应的稳定性；采用巯基末端修饰共价结合法将还原型捕获探针自组装于芯片金膜表面，探针浓度为1μM。
3.利用自行设计的锁式探针可以完全分辨靶序列单碱基突变，RCA可将SPR信号值(突变位点信号)放大10.0倍，突变识别率为5000：1(野生型与突变型靶序列的浓度比)。连接产物、RCA产物、以及RCA产物HhaI酶切电泳条带位置准确，明亮清晰。自行设计的捕获探针特异性好、各探针之间具有相同的长度和相近的Tm值(<1℃)，可使各探针在相同条件下杂交，适于传感器微阵列系统的多通道并行检测。检测最适液相靶序列杂交温度为45℃，芯片表面杂交温度为37℃，RCA扩增时间为30min。
4.针对5种临床常见结核杆菌耐药主要突变位点：异烟肼(INH)：KatG315(AGC→ACC), inhA-15(ACG→ATG)；利福平(PFP)：rpoB526(CAC→TAC)；乙胺丁醇(EMB)：embB306(ATG→GTG)；链霉素(SM)：rpsL43(AAG→AGG)，设计包含标签型锁式探针及相应捕获探针的探针组，并实现了芯片表面各突变位点的多重扩增检测，同一突变位点对突变型和野生型靶序列的SPR信号值差异明显(p <0.01)，各突变位点间交叉反应结果显示SPR信号值差异明显(p <0.01)。
5．建立的纳米金信号放大系统可有效提高检测的灵敏度，直径15nm的纳米金颗粒在pH7.5和8%工作浓度下可将SPR信号值(RCA放大后)放大2.1倍，与阴性和空白对照信号值比较有显著性差异(p <0.01)。对合成靶序列的检测检测限为5X10~(-12)M (59.1±7.1mo)，且在10-12M至10~(-8)M浓度范围内成线性相关(y=615.29x+1323.8, R2=0.9846)。
6.建立的液相target-primed RCA SPR生物传感器检测系统，可通过两步法在4h内完成对结核分枝杆菌群复合群(MTBC)和鸟型分支杆菌群复合群(MAC)的恒温(37℃)快速检测。SPR方法对临床标本的检测结果与菌种鉴定结果一致。最低检测限分别为MTBC：4.2X10~4CFU/mL (0.005ng/μL)，MAC：3.7x10~4CFU/mL (0.002ng/μL)；经特殊设计的锁式探针可使液相RCA反应与限制性内切酶反应同时进行，实现无标记的SPR实时动态监测RCA反应。
7.采用试剂盒法和加热碱裂解法对24例临床分离株标本和10例临床分泌物样本提取基因组DNA结果显示，碱裂解法操作简便，对临床分泌物标本和临床分离株均有较好的提取效果，可作为样本的前处理方法。SPR传感器方法检测34例临床标本，检测限为8.2pg/μL (65.7±8.5mo)临床标本基因组DNA，临床灵敏度和特异性与测序方法比较分别katG315：92%和80%；inhA-15：100%和100%；rpoB526：94.4%和100%；embB306：95%和100%；rpsL43：100%和100%。经Kappa检验两种方法检测结果具有较好的一致性性差异(p <0.05, Kappa>0.75)。
结论
1.新型恒温扩增型SPR生物传感器，改变了普通高灵敏性传感器检测对PCR方法的依赖。其稳定密闭的检测系统和简单低耗能的操作，有利于传感器的小型化和便携性。
2.自行设计的标签型锁式探针，不仅可在高背景干扰下识别突变靶序列，还可实现混合靶序列的多重连接反应。其包含的标签序列，可实现芯片表面的多重固相扩增反应，且反应条件一致，交叉反应小，增加了传感器微阵列的检测通量。
3.纳米金信号放大系统可显著增加SPR芯片表面的等离子共振强度，有效提高传感器的检测灵敏度，为SPR生物传感器的痕量物质检测提供了一种可靠的信号放大方法，在核酸非扩增快速检测及生物传感器领域有较大的应用潜力。
4.基于靶引物的液相RCA型SPR生物传感器检测系统，在保证特异性与敏感性的基础上，可快速恒温地实现对液相扩增反应的无标记实时动态监测，为核酸扩增检测提供了一条新思路。
5.构建的新型SPR生物传感器检测系统，可在4小时内同时检测五种临床常见MDRTB耐药突变位点，检测方法具有灵敏、特异、操作简单、快速的特点。与传统基因芯片相比，极大降低了技术、设备要求和检测成本。为临床结核杆菌耐药的恒温快速检测乃至其他基因检测提供一种新方法新思路，有望弥补现有常规检测方法存在的不足，满足临床快速诊断的需求，具有广阔的应用前景。
Background
Due to the increase of population number and mobility, non-standard drug-use, as wellas the HIV/AIDS pandemic, a steady drug-resistant increase of M.tuberculosis hasthreatened tuberculosis (TB) control world-widely, despite advancements in the diagnosisand treatment of TB over the years. The multidrug-resistant strains were defined as beingresistant to at least isoniazid and rifampin. The emergence and spread of multidrug-resistantMycobacterium tuberculosis (MDRTB) even extensively drug resistant tuberculosis(XDRTB) posed an upward trend and represented an increasing public health problem.Conventional culture-based bacterial identification method or sequence analysis canprovide definitive results, however, it is time-consuming, laborious and expensive.Therefore, the development of simple, sensitive and low-cost method is important for therapid detection of drug-resistance. It is a timely and effective way to diagnose and manageTB patients, especially MDRTB infected.
Because of high sensitivity of optical transducer, convenient operation and real-timemonitoring, surface plasmon resonance (SPR) biosensors have been adopted in numerousstudies on variety of bimolecular interaction including DNA, RNA, protein and bacteriumbased on different recognition elements. In a SPR biosensor assay, the change of therefractive index on the gold film correlates with the amount of analyte bound to the film. Inmost of SPR-based DNA detection with high sensitivity, nucleic acid amplification isadopted to amplify analyte mass, especially polymerase chain reaction (PCR). However, thenonspecific amplification from complicated thermal circling step, non-compatibility withon-chip amplification and increase of detection cost are still problems to be considered.
Focusing on the development of isothermal amplification methods, RCA has shown itsattraction due to the specificity and multiplexing besides simplicity. The L-RCA by ligase relies on base pairing principle which requires perfect complementarity on the ligation nick.It not only forbids the mismatch but also has a low occurrence of false positive results whencompared to PCR. When used as a signal amplification method, this property enabled highmultiplexing without interference, direct amplification on solid phase and precise productlocalization. Moreover, RCA amplifies the circular PLP only, without accumulation oftarget templates over time, which minimizes the risk of contamination and the potentialbiohazard.
In this study, we developed a SPR DNA biosensor method combined withsurface-anchored RCA to isothermally detect multiple point mutations associated withdrug-resistance in MDRTB. The Au nanoparticle signal amplification was adopted tofurther improve the sensitivity. The sensitivity and specificity of the SPR detection systemwere evaluated and the application was tested in real clinical samples. Besides, we alsodeveloped a SPR real-time detection method by target-primed RCA to discriminate TBbacilli from nontuberculous mycobacteria. This study lays the groundwork for applying theSPR biosensor for isothermal and rapid detection of resisitence associated mutations inM.tuberculosis.
Methods
1.The solid-phase RCA mode was contrasted by using magnetic beads. Theamino-modified oligonucleotide probe (capture probe) was immobilized on the surface ofthe carboxyl-modified magnetic beads through covalent coupling principle to form sensingmembrane. The optimum detection procedure and reaction condition were investigated. Theliquid phase RCA reaction condition was also investigated by the fluorescence quantitativemethod with SRBR Green II dye.
2.Based on the previous study, the SPR biosensor platform was constructed withimprovements to increase the detection channel, improve temperature control, flow controland analyte circulating ways. The control software UMPHO-A600was designed tointerface with the SPR detector. The stability and reproducibility were evaluated. Theself-assembly of the sensor chip was improved to prepare microarrays.
3.The sequences of five clinically meaningful mutations in MDRTB were searched forin the NCBI database. The padlock probe (PLP) containing a specific tag-sequence, ageneral sequence, and a target-specific region on each detection arm, was designed with Primer Premier5.0software. The capture probe containing tag-sequence with consistencyand specificity was designed by Array Designer4.0. The second structure prediction of thecircular PLP was also analyzed.
4.Combination of Au nanoparticle signal amplification with the surface-anchored RCAresulted in cascade signal amplification on the chip surface. The experimental conditionsfor the detection system were also optimized. The synthetic oligonucleotides were used toevaluate the sensitivity and specificity of the detection system.
5.According to the sequence of ITS in16s-23s rRNA gene found in GenBank, PLPwas designed to contain the restriction cutting site. The target-primed RCA was establishedto discriminate M.tuberculosis from NTM with the SPR biosensor. The dynamic real-timemonitoring of liquid-phase RCA with label-free manner was achieved.
6.The detection system was demonstrated by targeting five resistance associatedmutations in MDRTB from34cased clinical samples. The sequencing was made asreference. The sensitivity and specificity of the developed SPR biosensor system formultiplex mutation detections of clinical samples were tested.
Results
1.The solid-phase RCA system was achieved on the carrier of magnetic beads and theamplification way was determined as ligation in solution combined with surfaceamplification. The optimum solid-phase RCA reaction condition was determined as0.5U/μL Phi29DNA polymerase，0.2μg/μL BSA，5%DMSO，1mM dNTPs. Theelectrophoresis verification showed that the circular PLP could be generated only in thepresence of mutant target to cause the following surface RCA reaction, whereas no reactionhappened.
2.The sensor chip was the gold film prim with the size of20mm x28.6mm. Thedetection well was eight-channel tandem. The temperature was controlled with an accuracyof±0.1℃at25~60℃. The flow rate could be changed from5μL/min to2000μL/min withan accuracy of1μL/min. the detection well was in negative pressure to form a sealcondition to maintain the stability of the reaction during reaction. There was scarcelyinterference between channels to achieve the array detection. The thiol group modifiedcapture probes were self-assembled on the chip surface through the covalent binding withthe concentration of1μM.
3.The designed PLP can distinguish the target with single-base mismatch. RCA canamplify the SPR signal for almost10-fold. The positive mutation detection is achieved witha wild-type to mutant ratio of5000:1. The electrophoresis bands of ligation products, RCAproducts and restriction enzyme products of RCA were accurate and clear. The designedcapture probes with the similar length and Tm values (<1℃) can react under the samecondition, but also owned the high specificity. The optimum hybridization temperature inligation was45℃, while on the chip surface was37℃. The RCA duration time was30min.
4.The probe sets were designed to target five common clinical resistance associatedmutation site, including isoniazid (INH): KatG315(AGC→ACC), inhA-15(ACG→ATG); rifampicin (PFP): rpoB526(CAC→TAC); ethambutol (EMB): embB306(ATG→GTG); streptomycin (SM): rpsL43(AAG→AGG). The multiplex amplification on chipsurface was achieved. The SPR signal difference between mutant and wild-type target wassignificant (p <0.01). The cross reaction result showed that the SPR signal differencebetween each mutation site was significant (p <0.01).
5.The Au nanoparticle signal amplification system could effectively improve thesensitivity for2.1-fold, with15nm diameter, pH7.5and8%of concentration, whichshowed significant difference (p <0.01) compared to the negative control and blank control.The detection limit was5x10-12M synthetic oligonucleotides, and a linear correlation (y=615.29x+1323.8, R2=0.9846) was found within the concentration range of10-12M to10-8M.
6.The developed target-primed RCA SPR biosensor system to isothermally (37℃)and rapidly (within4h) detect MTBC and MAC could be performed by only two steps. Theresults of clinical samples from SPR method and bacteria identification were consistence.The detection limit for MTBC was4.2X104CFU/mL (0.005ng/μL), and MAC was3.7x104CFU/mL (0.002ng/μL), respectively. The specially designed PLP could make theliquid-phase RCA and digestion reaction perform simultaneously, which achieve thedynamic real-time monitoring of RCA by SPR biosensor.
7.The heated alkali lysis method was more simple, cost-effective and sensitive toextract genomic DNA in clinical samples. Compared to the sequencing method, thesensitivity and specificity of the SPR detection were92%and80%, respectively, for katG 315,100%and100%, respectively, for inhA-15,94.4%and100%, respectively, for rpoB526,95%and100%, respectively, for embB306,100%and100%, respectively, for rpsL43(Table2). According to the Kappa test,(p <0.05, Kappa>0.75) there exists significantconsistency between the two methods.
Conclusions
1.The developed isothermal amplification based SPR biosensor changed the detectionmethod of PCR-dependent traditional biosensors. The sealed and stable detection systemwith convenience and low power consumption was favor of miniaturization and portabilityapplication.
2.The designed PLP had excellent capability of detecting the low-abundance mutationin the large background of wild targets, but also achieved the multiplex ligation with mixedtargets. The tag-sequence achieved the multiplex surface-anchored RCA with same reactioncondition and no interference, improved the multiplexing capabilities of the biosensor arrayand simplified the operation.
3.Au nanoparticle signal amplification system could significantly increase the plasmonoscillations with a resonant frequency, thus improve the signal/noise ratio and thesensitivity. This system provided a reliable signal amplification method to detect traceanalyte, and hold great promise in scientific and clinical diagnostic application.
4.The target-primed RCA SPR biosensor system achieved dynamically real-timemonitor the liquid amplification with label-free manners, which provided a rapid andisothermal detection method with specificity and sensitivity.
5. Compared with the sequencing method, the developed SPR biosensor detectionsystem has good sensitivity and specificity in the detection of clinical samples, whichindicated that the novel SPR biosensor based on isothermal amplification would be apotential clinical diagnostic method for rapid detection of multiple resistance associatedmutation or other mutations associated disease in clinical samples
引文
[1] Organization WH: Global Tuberculosis Report2012.2012.
[2] Tang S, Zhang Q, Yu J, Liu Y, Sha W, Sun H, Fan L, Gu J, Hao X, Yao L: Extensivelydrug-resistant tuberculosis, China. Emerging infectious diseases2011,17:558.
[3] Diedrich CR, Flynn JL: HIV-1/mycobacterium tuberculosis coinfection immunology:how does HIV-1exacerbate tuberculosis? Infect Immun2011,79:1407-1417.
[4] Balabanova Y, Tchernyshev V, Tsigankov I, Maximova S, Mikheeva N, FedyukovitchL, Kuznetsov S, Fedorin I, Drobniewski F: Analysis of undiagnosedtuberculosis-related deaths identified at post-mortem among HIV-infected patients inRussia: a descriptive study. BMC infectious diseases2011,11:276.
[5] Weyer K, Carai S, Nunn P: Viewpoint TB Diagnostics: What Does the World ReallyNeed? The Journal of infectious diseases2011,204Suppl4:S1196-1202.
[6] Morisset D, Stebih D, Cankar K, Zel J, Gruden K: Alternative DNA amplificationmethods to PCR and their application in GMO detection: a review. European FoodResearch and Technology2008,227:1287-1297.
[7] Lee MF, Chen YH, Hsu HJ, Peng CF: One-tube loop-mediated isothermalamplification combined with restriction endonuclease digestion and ELISA forcolorimetric detection of resistance to isoniazid, ethambutol and streptomycin inMycobacterium tuberculosis isolates. Journal of microbiological methods2010,83:53-58.
[8] Zhang D, Wu J, Ye F, Feng T, Lee I, Yin B: Amplification of circularizable probes forthe detection of target nucleic acids and proteins. Clinica chimica acta; internationaljournal of clinical chemistry2006,363:61-70.
[9] Schweitzer B, Kingsmore S: Combining nucleic acid amplification and detection.Curr Opin Biotech2001,12:21–27.
[10] Nallur G, Luo C, Fang L, Cooley S, Dave V, Lambert J, Kukanskis K, Kingsmore S,Lasken R, Schweitzer. B: single amplification by rolling circle amplification on DNAmicroarrays. Nucleic acids research2001,29:e118.
[11] Banér J, Nilsson M, Isaksson A, Mendel-Hartvig M, Antson D, Ulf Landegren U:More keys to padlock probes mechanisms for high-throughput nucleic acid analysis.Curr Opin Biotech2001,12:11–15.
[12] Banér J, Nilsson M, Mendel-Hartvig M, Landegren U: Signal amplification ofpadlock probes by rolling circle replication. Nucleic acids research1998,26:5073-5078.
[13] Demidov VV: Rolling-circle amplification in DNA diagnostic: the power of simplicity.Expert Rev Mol Dtagn2002,2:542-548.
[14] Homola J: Surface Plasmon Resonance Sensors for Detection of Chemical andbiological species. Chem Rev2008,108462-493.
[15] Bailey RC, Qavi AJ, Washburn AL, Byeon JY: Label-free technologies forquantitative multiparameter biological analysis. Analytical and bioanalyticalchemistry2009,394:121-135.
[16] Gusev Y, Sparkowski J, Raghunathan A, Ferguson Jr H, Montano J, Bogdan N,Schweitzer B, Wiltshire S, Kingsmore SF, Maltzman W: Rolling circle amplification:a new approach to increase sensitivity for immunohistochemistry and flow cytometry.The American journal of pathology2001,159:63-69.
[17] Blanco L, Bernad A, Martin G, Garmendia C, Salas M: Highly Efficient DNASynthesis by the Phage Φ29DNA Polymerase. Biological Chemistry1988264:8935-8940.
[18] Liu L, Tang Z, Wang K, Tan W, Li J, Guo Q, Meng X, Ma C: Using molecular beaconto monitor activity of E. coli DNA ligase. Analyst2005,130:350-357.
[19] Tong J, Cao W, Barany F: Biochemical properties of a high fidelity DNA ligase fromThermus species AK16D. Nucleic acids research1999,27:788-794.
[20] Yao C, Zhu T, Tang J, Wu R, Chen Q, Chen M, Zhang B, Huang J, Fu W:Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor.Biosensors&bioelectronics2008,23:879-885.
[21] Xia H, Wang F, Huang Q, Huang J, Chen M, Wang J, Yao C, Chen Q, Cai G, Fu W:Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance NucleicAcid Biosensor Array Using Au Nanoparticle Signal Amplification. Sensors2008,8:6453-6470.
[22] Wang J, Luo Y, Zhang B, Chen M, Huang J, Zhang K, Gao W, Fu W, Jiang T, Liao P:Rapid label-free identification of mixed bacterial infections by surface plasmonresonance. Journal of translational medicine2011,9:85.
[23] Lopez CA, Daaboul GG, Vedula RS, Ozkumur E, Bergstein DA, Geisbert TW,Fawcett HE, Goldberg BB, Connor JH, Unlu MS: Label-free multiplexed virusdetection using spectral reflectance imaging. Biosensors&bioelectronics2011,26:3432-3437.
[24] Kim YS, Yi SY, Kim J, Kim M, Kim CS, Chung BH, Kim JS: Novel application ofsurface plasmon resonance biosensor chips for measurement of advanced glycationend products in serum of Zucker diabetic fatty rats. Biosensors&bioelectronics2009,25:248-252.
[25] Mitchell JS, Lowe TE: Ultrasensitive detection of testosterone using conjugate linkertechnology in a nanoparticle-enhanced surface plasmon resonance biosensor.Biosensors&bioelectronics2009,24:2177-2183.
[26] Milkani E, Morais S, Lambert CR, McGimpsey WG: Detection of oligonucleotidesystematic mismatches with a surface plasmon resonance sensor. Biosensors&bioelectronics2010,25:1217-1220.
[27] Mannelli I, Courtois V, Lecaruyer P, Roger G, Millot MC, Goossens M, Canva M:Surface plasmon resonance imaging (SPRI) system and real-time monitoring of DNAbiochip for human genetic mutation diagnosis of DNA amplified samples. SensorActuat B-Chem2006,119:583-591.
[28] Florschütz K, Schr ter A, Schmieder S, Chen W, Schweizer P, Sonntag F, Danz N,Baronian K, Kunze G:‘Phytochip’–on-chip detection of phytopathogenic RNAviruses by a new surface plasmon resonance platform. Journal of virological methods2013.
[29] Nedelkov D, Nelson RW: Surface plasmon resonance mass spectrometry: recentprogress and outlooks. TRENDS in Biotechnology2003,21:301-305.
[30] Raitman OA, Katz E, Bückmann AF, Willner I: Integration of polyaniline/poly(acrylic acid) films and redox enzymes on electrode supports: an in situelectrochemical/surface plasmon resonance study of the bioelectrocatalyzed oxidationof glucose or lactate in the integrated bioelectrocatalytic systems. Journal of theAmerican Chemical Society2002,124:6487-6496.
[31] Hu WP, Chen SJ, Huang KT, Hsu JH, Chen WY, Chang GL, Lai KA: A novelultrahigh-resolution surface plasmon resonance biosensor with an Aunanocluster-embedded dielectric film. Biosensors&bioelectronics2004,19:1465-1471.
[32] Wood R: XLII. On a remarkable case of uneven distribution of light in a diffractiongrating spectrum. The London, Edinburgh, and Dublin Philosophical Magazine andJournal of Science1902,4:396-402.
[33] Ritchie R: Plasma losses by fast electrons in thin films. Physical Review1957,106:874.
[34] Stern E, Ferrell R: Surface plasma oscillations of a degenerate electron gas. PhysicalReview1960,120:130.
[35] Kretschmann E: The determination of the optical constants of metals by excitation ofsurface plasmons. z Phys1971,241:313-324.
[36] Liedberg B, Nylander C, Lunstr m I: Surface plasmon resonance for gas detectionand biosensing. Sensors and actuators1983,4:299-304.
[37] Dell’Atti D, Tombelli S, Minunni M, Mascini M: Detection of clinically relevantpoint mutations by a novel piezoelectric biosensor. Biosensors and Bioelectronics2006,21:1876-1879.
[38] Chen M, Liu M, Yu L, Cai G, Chen Q, Wu R, Wang F, Zhang B, Jiang T, Fu W:Construction of a novel peptide nucleic acid piezoelectric gene sensor microarraydetection system. Journal of nanoscience and nanotechnology2005,5:1266-1272.
[39] D'Agata R, Corradin R, Ferretti C, Zanoli L, Gatti M, Marchelli R, Spoto G:Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhancedsurface plasmon resonance imaging. Biosensors&bioelectronics2010,25:2095-2100.
[40][Wang J, Zhou HS: Aptamer-based Au nanoparticles-enhanced surface plasmonresonance detection of small molecules. Analytical chemistry2008,80:7174-7178.
[41] Zynio SA, Samoylov AV, Surovtseva ER, Mirsky VM, Shirshov YM: Bimetalliclayers increase sensitivity of affinity sensors based on surface plasmon resonance.Sensors2002,2:62-70.
[42] Hanken DG, Jordan CE, Frey BL, Corn RM: Surface plasmon resonancemeasurements of ultrathin organic films at electrode surfaces. ElectroanalyticalChemistry1998,20:141-226.
[43] Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA: Selective colorimetricdetection of polynucleotides based on the distance-dependent optical properties ofgold nanoparticles. Science1997,277:1078-1081.
[44] Zhang S, Wu Z, Shen G, Yu R: A label-free strategy for SNP detection with highfidelity and sensitivity based on ligation-rolling circle amplification and intercalatingof methylene blue. Biosensors&bioelectronics2009,24:3201-3207.
[45] Yao C, Xiang Y, Deng K, Xia H, Fu W: Sensitive and specific HBV genomic DNAdetection using RCA-based QCM biosensor. Sensor Actuat B-Chem2013.
[46] Lucarelli F, Marrazza G, Turner AP, Mascini M: Carbon and gold electrodes aselectrochemical transducers for DNA hybridisation sensors. Biosensors andBioelectronics2004,19:515-530.
[47] Lai RY, Lagally ET, Lee S-H, Soh H, Plaxco KW, Heeger AJ: Rapid,sequence-specific detection of unpurified PCR amplicons via a reusable,electrochemical sensor. Proceedings of the National Academy of Sciences of theUnited States of America2006,103:4017-4021.
[48] Su Q, Xing D, Zhou X: Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation.Biosensors&bioelectronics2010,25:1615-1621.
[49] Nilsson M, Gullberg M, Dahi F, Szhuai K, Raap AK: Real-time mmonitoring ofrolling-circle amplification using a modificated molecular beacon design. Nucleicacids research2002,30:e66.
[50] McCarthy EL, Bickerstaff LE, da Cunha MP, Millard PJ: Nucleic acid sensing byregenerable surface-associated isothermal rolling circle amplification. Biosensors&bioelectronics2007,22:1236-1244.
[51] Chen Q, Bian Z, Chen M, Hua X, Yao C, Xia H, Kuang H, Zhang X, Huang J, Cai G,Fu W: Real-time monitoring of the strand displacement amplification (SDA) ofhuman cytomegalovirus by a new SDA-piezoelectric DNA sensor system. Biosensors&bioelectronics2009,24:3412-3418.
[52] Wartell RM, Reznikoff WS: Cloning DNA restriction endonuclease fragments withprotruding single-stranded ends. Gene1990,9:307-319.
[53] Dean FB, Nelson JR, Giesler TL, Lasken RS: Rapid amplification of plasmid andphage DNA using phi29DNA polymerase and multiply-primed rolling circleamplification. Genome research2001,11:1095-1099.
[54] Sharma K, Sharma A, Modi M, Singh G, Kaur H, Varma S, Sharma M: PCR detectionof co-infection with Mycobacterium tuberculosis and Mycobacterium avium in AIDSpatients with meningitis. J Med Microbiol2012,61:1789-1791.
[55] Larsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M: In situgenotyping individual DNA molecules by target-primed rolling-circle amplification ofpadlock probes. Nature methods2004,1:227-232.
[56] Andersson C, Henriksson S, Magnusson KE, Nilsson M, Mirazimi A: In situ rollingcircle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV)complementary and viral RNA. Virology2012,426:87-92.
[57] Henriksson S, Blomstrom AL, Fuxler L, Fossum C, Berg M, Nilsson M: Developmentof an in situ assay for simultaneous detection of the genomic and replicative form ofPCV2using padlock probes and rolling circle amplification. Virology journal2011,8:37.
[58] Motre A, Kong R, Li Y: Improving isothermal DNA amplification speed for the rapiddetection of Mycobacterium tuberculosis. Journal of microbiological methods2011,84:343-345.
[59] Torres-Chavolla E, Alocilja EC: Nanoparticle based DNA biosensor for tuberculosisdetection using thermophilic helicase-dependent isothermal amplification. Biosensors&bioelectronics2011,26:4614-4618.
[60] Long Y, Zhou X, Xing D: Sensitive and isothermal electrochemiluminescencegene-sensing of Listeria monocytogenes with hyperbranching rolling circleamplification technology. Biosensors&bioelectronics2011,26:2897-2904.
[61] Aryan E, Makvandi M, Farajzadeh A, Huygen K, Bifani P, Mousavi SL, Fateh A,Jelodar A, Gouya MM, Romano M: A novel and more sensitive loop-mediatedisothermal amplification assay targeting IS6110for detection of Mycobacteriumtuberculosis complex. Microbiological research2010,165:211-220.
[62] Prabhakar N, Arora K, Arya SK, Solanki PR, Iwamoto M, Singh H, Malhotra BD:Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance.Analyst2008,133:1587-1592.
[63] Ramaswamy S, Musser JM: Molecular genetic basis of antimicrobial agent resistancein Mycobacterium tuberculosis:1998update. Tubercle and Lung Disease1998,79:3–29.
[64] Tracevska T, Jansone I, Nodieva A, Marga O, Skenders G, Baumanis V:Characterisation of rpsL, rrs and embB mutations associated withstreptomycin and ethambutol resistance in Mycobacterium tuberculosis.Research in microbiology2004,155:830-834.
[65] Heym B, Alzari PM, Honore N, Cole ST: Missense mutations in the catalase‐peroxidase gene, katG, are associated with isoniazid resistance in Mycobacteriumtuberculosis. Molecular microbiology1995,15:235-245.
[66] Miller LP, Crawford JT, Shinnick TM: The rpoB gene of Mycobacterium tuberculosis.Antimicrobial agents and chemotherapy1994,38:805-811.
[67] Sreevatsan S, Stockbauer KE, Pan X, Kreiswirth BN, Moghazeh SL, Jacobs W,Telenti A, Musser JM: Ethambutol resistance in Mycobacterium tuberculosis: criticalrole of embB mutations. Antimicrobial agents and chemotherapy1997,41:1677-1681.
[68] Finken M, Kirschner P, Meier A, Wrede A, B ttger EC: Molecular basis ofstreptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomalprotein S12gene and point mutations within a functional16S ribosomal RNApseudoknot. Molecular microbiology1993,9:1239-1246.
[69] Haas WH, Schilke K, Brand J, Amthor B, Weyer K, Fourie PB, Bretzel G, Sticht-GrohV, Bremer HJ: Molecular analysis of katG gene mutations in strains ofMycobacterium tuberculosis complex from Africa. Antimicrobial agents andchemotherapy1997,41:1601-1603.
[70] Kiepiela P, Bishop K, Smith A, Roux L, York D: Genomic mutations in the katG, inhAand aphC genes are useful for the prediction of isoniazid resistance in Mycobacteriumtuberculosis isolates from Kwazulu Natal, South Africa. Tubercle and lung disease:the official journal of the International Union against Tuberculosis and Lung Disease2000,80:47.
[71] Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, Collins D,de Lisle G, Jacobs W: inhA, a gene encoding a target for isoniazid and ethionamide inMycobacterium tuberculosis. Science1994,263:227-230.
[72] Mikhailovich V, Lapa S, Gryadunov D, Sobolev A, Strizhkov B, Chernyh N,Skotnikova O, Irtuganova O, Moroz A, Litvinov V: Identification of rifampin-resistant Mycobacterium tuberculosis strains by hybridization, PCR, and ligasedetection reaction on oligonucleotide microchips. Journal of clinical microbiol-ogy2001,39:2531-2540.
[73] Ahmad S, Mokaddas E: The occurrence of rare rpoB mutations inrifampicin-resistant clinical Mycobacterium tuberculosis isolates fromKuwait. International journal of antimicrobial agents2005,26:205-212.
[74] Plinke C, Rüsch-Gerdes S, Niemann S: Significance of mutations in embB codon306for prediction of ethambutol resistance in clinical Mycobacterium tuberculosisisolates. Antimicrobial agents and chemotherapy2006,50:1900-1902.
[75] Dobner P, Bretzel G, Rüsch-Gerdes S, Feldmann K, Rifai M, L scher T, Rinder H:Geographic variation of the predictive values of genomic mutations associated withstreptomycin resistance in Mycobacterium tuberculosis. Molecular andcellular probes1997,11:123-126.
[76] Yao C, Zhu T, Li Y, Zhang L, Zhang B, Huang J, Fu W: Detection of rpoB, katG andinhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqingas determined by microarray Clin Microbiol Infect2010,16:1639–1643.
[77] Telenti A, Imboden P, Marchesi F, Matter L, Schopfer K, Bodmer T, Lowrie D,Colston M, Cole S: Detection of rifampicin-resistance mutations in Mycobacteriumtuberculosis. The Lancet1993,341:647-651.
[78] Caoili JC, Mayorova A, Sikes D, Hickman L, Plikaytis BB, Shinnick TM: Evaluationof the TB-Biochip oligonucleotide microarray system for rapid detection of rifampinresistance in Mycobacterium tuberculosis. Journal of clinical microbiology2006,44:2378-2381.
[79] Brossier F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W: Performance of thegenotype MTBDR line probe assay for detection of resistance to rifampin andisoniazid in strains of Mycobacterium tuberculosis with low-and high-level resistance.Journal of clinical microbiology2006,44:3659-3664.
[80] Duman M, Piskin E: Detection of Mycobacterium tuberculosis complex andMycobacterium gordonae on the same portable surface plasmon resonance sensor.Biosensors&bioelectronics2010,26:908-912.
[81] Marín M, De Viedma DG, Ruíz-Serrano MJ, Bouza E: Rapid direct detection ofmultiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosisin respiratory samples by real-time PCR. Antimicrobial agents and chemotherapy2004,48:4293-4300.
[82] Pai M, Kalantri S, Dheda K: New tools and emerging technologies for the diagnosisof tuberculosis: part II. Active tuberculosis and drug resistance. Expert review ofmolecular diagnostics2006,6:423-432.
[83] Hatch A, Takeshi S, Misasi J, Smith LC: Rolling circle amplification of DNAimmobilized on solid surfaces and its application to multiplex mutation detetion.Genet Anal-Biomole199915:35-40.
[84] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, KingsmoreSF, Lizardi PM, Ward DC: Immunoassays with rolling circle DNA amplification: aversatile platform for ultrasensitive antigen detection. Proceedings of the NationalAcademy of Sciences2000,97:10113-10119.
[85] Thomas DC, Nardone GA, Randall SK: Amplification of padlock probes for DNAdiagnostics by cascade rolling circle amplification or the polymerase chain reaction.Archives of pathology&laboratory medicine1999,123:1170-1176.
[86] Wilkomson D: Third wave technologies' invader assays for nucleic acid detection.The Scientist1993,13:16-18.
[87] Hardenbol p, Baner J, Jain M, Nilsson M, Namsaraev E: Multiplexed genotyping withsequence-tagged molecular inversion probes. Nature biotechnology2003,21:673-678.
[88] Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Kwiatkowski RW,Sander TJ, de Arruda M, Arco DA: Polymorphism identification and quantitativedetection of genomic DNA by invasive cleavage of oligonucleotide probes. Naturebiotechnology1999,17:292-296.
[89] ALLAWI HT, DAHLBERG JE, OLSON S, LUND E, OLSON M, MA W-P, TAKOVAT, NERI BP, LYAMICHEV VI: Quantitation of microRNAs using a modified Invaderassay. Rna2004,10:1153-1161.
[90] Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C-a, Horn T, Ahle D, DetmerJ, Shen L-P: A branched DNA signal amplification assay for quantification of nucleicacid targets below100molecules/ml. Nucleic acids research1997,25:2979-2984.
[91] Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS,Perrillo RP: Quantitation of HBV DNA in human serum using a branched DNA(bDNA) signal amplification assay. American journal of clinical pathology1995,104:537.
[92] Pachl C, Todd JA, Kern DG, Sheridan PJ, Fong S-J, Stempien M, Hoo B, Besemer D,Yeghiazarian T, Irvine B: Rapid and precise quantification of HIV-1RNA in plasmausing a branched DNA signal amplification assay. JAIDS Journal of AcquiredImmune Deficiency Syndromes1995,8:446&hyhen.
[93] Zhang DY, Winfree E: Robustness and modularity properties of a non-covalent DNAcatalytic reaction. Nucleic acids research2010,38:4182-4197.
[94] Choi HM, Chang JY, Trinh LA, Padilla JE, Fraser SE, Pierce NA: Programmable insitu amplification for multiplexed imaging of mRNA expression. Naturebiotechnology2010,28:1208-1212.
[95] Huang J, Wu Y, Chen Y, Zhu Z, Yang X, Yang CJ, Wang K, Tan W: Pyrene‐ExcimerProbes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acidsin Complex Biological Fluids. Angewandte Chemie International Edition2011,50:401-404.
[96] Zhang B, Liu B, Tang D, Niessner R, Chen G, Knopp D: DNA-Based HybridizationChain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection ofProteins. Analytical chemistry2012,84:5392-5399.
[97] Wang C, Zhou H, Zhu W, Li H, Jiang J, Shen G, Yu R: Ultrasensitive electrochemicalDNA detection based on dual amplification of circular strand-displacementpolymerase reaction and hybridization chain reaction. Biosensors and Bioelectronics2013.
[98] Dirks RM, Pierce NA: Triggered amplification by hybridization chain reaction.Proceedings of the National Academy of Sciences of the United States of America2004,101:15275-15278.
[99] Reichert J, Csáki A, K hler JM, Fritzsche W: Chip-based optical detection of DNAhybridization by means of nanobead labeling. Analytical chemistry2000,72:6025-6029.
[100] Cao YC, Jin R, Mirkin CA: Nanoparticles with Raman spectroscopic fingerprints forDNA and RNA detection. Science2002,297:1536-1540.
[101] Taton TA, Lu G, Mirkin CA: Two-color labeling of oligonucleotide arrays viasize-selective scattering of nanoparticle probes. Journal of the American ChemicalSociety2001,123:5164.
[102] Petryayeva E, Krull UJ: Localized surface plasmon resonance: nanostructures,bioassays and biosensing--a review. Analytica chimica acta2011,706:8-24.
[103] He L, Musick MD, Nicewarner SR, Salinas FG, Benkovic SJ, Natan MJ, Keating CD:Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection ofDNA hybridization. Journal of the American Chemical Society2000,122:9071-9077.
[104] Hutter E, Pileni M-P: Detection of DNA hybridization by gold nanoparticle enhancedtransmission surface plasmon resonance spectroscopy. The Journal of PhysicalChemistry B2003,107:6497-6499.
[105] Mao X, Yang L, Su XL, Li Y: A nanoparticle amplification based quartz crystalmicrobalance DNA sensor for detection of Escherichia coli O157:H7. Biosensors&bioelectronics2006,21:1178-1185.
[106] Chen SH, Wu VC, Chuang YC, Lin CS: Using oligonucleotide-functionalized Aunanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor.Journal of microbiological methods2008,73:7-17.
[107] Liu T, Tang Ja, Jiang L: Sensitivity enhancement of DNA sensors by nanogold surfacemodification. Biochemical and biophysical research communications2002,295:14-16.
[108] Patolsky F, Ranjit KT, Lichtenstein A, Willner I: Dendritic amplification of DNAanalysis by oligonucleotide-functionalized Au-nanoparticles. Chem Commun2000:1025-1026.
[109] Weizmann Y, Patolsky F, Willner I: Amplified detection of DNA and analysis ofsingle-base mismatches by the catalyzed deposition of gold on Au-nanoparticles.Analyst2001,126:1502-1504.
[110] M ller R, Csaki A, K hler J, Fritzsche W: Electrical classification of theconcentration of bioconjugated metal colloids after surface adsorption and silverenhancement. Langmuir2001,17:5426-5430.
[111] Park S-J, Taton TA, Mirkin CA: Array-based electrical detection of DNA withnanoparticle probes. Science2002,295:1503-1506.
[112] Cai H, Xu C, He P, Fang Y: Colloid Au-enhanced DNA immobilization for theelectrochemical detection of sequence-specific DNA. Journal of electroanalyticalchemistry2001,510:78-85.
[113] Wang J, Xu D, Kawde A-N, Polsky R: Metal nanoparticle-based electrochemicalstripping potentiometric detection of DNA hybridization. Analytical chemistry2001,73:5576-5581.
[114] Teresa. Torre, Mattias. Stromberg, Camilla. Russell, Jenny. Goransson, Mats. Nilsson,Peter. Svedlindh, Maria. Stromme: Investigation of Immobilization of FunctionalizedMagnetic Nanobeads in Rolling Circle Amplified DNA Coils. J. Phys. Chem. B2010,114:3707-3713.
[1] Hatch A, Takeshi S, Misasi J, Smith LC: Rolling circle amplification of DNAimmobilized on solid surfaces and its application to multiplex mutation detetion.Genet Anal-Biomole199915:35-40.
[2] Banér J, Nilsson M, Isaksson A, Mendel-Hartvig M, Antson D, Ulf Landegren U:More keys to padlock probes mechanisms for high-throughput nucleic acid analysis.Curr Opin Biotech2001,12:11–15.
[3] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, KingsmoreSF, Lizardi PM, Ward DC: Immunoassays with rolling circle DNA amplification: aversatile platform for ultrasensitive antigen detection. Proceedings of the NationalAcademy of Sciences2000,97:10113-10119.
[4] Thomas DC, Nardone GA, Randall SK: Amplification of padlock probes for DNAdiagnostics by cascade rolling circle amplification or the polymerase chain reaction.Archives of pathology&laboratory medicine1999,123:1170-1176.
[5] Wilkomson D: Third wave technologies' invader assays for nucleic acid detection.The Scientist1993,13:16-18.
[6] Hardenbol p, Baner J, Jain M, Nilsson M, Namsaraev E: Multiplexed genotyping withsequence-tagged molecular inversion probes. Nature biotechnology2003,21:673-678.
[7] Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Kwiatkowski RW,Sander TJ, de Arruda M, Arco DA: Polymorphism identification and quantitativedetection of genomic DNA by invasive cleavage of oligonucleotide probes. Naturebiotechnology1999,17:292-296.
[8] ALLAWI HT, DAHLBERG JE, OLSON S, LUND E, OLSON M, MA W-P, TAKOVAT, NERI BP, LYAMICHEV VI: Quantitation of microRNAs using a modified Invaderassay. Rna2004,10:1153-1161.
[9] Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C-a, Horn T, Ahle D, DetmerJ, Shen L-P: A branched DNA signal amplification assay for quantification of nucleicacid targets below100molecules/ml. Nucleic acids research1997,25:2979-2984.
[10] Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS,Perrillo RP: Quantitation of HBV DNA in human serum using a branched DNA(bDNA) signal amplification assay. American journal of clinical pathology1995,104:537.
[11] Pachl C, Todd JA, Kern DG, Sheridan PJ, Fong S-J, Stempien M, Hoo B, Besemer D,Yeghiazarian T, Irvine B: Rapid and precise quantification of HIV-1RNA in plasmausing a branched DNA signal amplification assay. JAIDS Journal of AcquiredImmune Deficiency Syndromes1995,8:446&hyhen.
[12] Zhang DY, Winfree E: Robustness and modularity properties of a non-covalent DNAcatalytic reaction. Nucleic acids research2010,38:4182-4197.
[13] Choi HM, Chang JY, Trinh LA, Padilla JE, Fraser SE, Pierce NA: Programmable insitu amplification for multiplexed imaging of mRNA expression. Nature biotechn-ology2010,28:1208-1212.
[14] Huang J, Wu Y, Chen Y, Zhu Z, Yang X, Yang CJ, Wang K, Tan W: Pyrene‐ExcimerProbes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acidsin Complex Biological Fluids. Angewandte Chemie International Edition2011,50:401-404.
[15] Zhang B, Liu B, Tang D, Niessner R, Chen G, Knopp D: DNA-Based HybridizationChain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection ofProteins. Analytical chemistry2012,84:5392-5399.
[16] Wang C, Zhou H, Zhu W, Li H, Jiang J, Shen G, Yu R: Ultrasensitive electrochemicalDNA detection based on dual amplification of circular strand-displacementpolymerase reaction and hybridization chain reaction. Biosensors and Bioelectronics2013.
[17] Dirks RM, Pierce NA: Triggered amplification by hybridization chain reaction.Proceedings of the National Academy of Sciences of the United States of America2004,101:15275-15278.
[18] Reichert J, Csáki A, K hler JM, Fritzsche W: Chip-based optical detection of DNAhybridization by means of nanobead labeling. Analytical chemistry2000,72:6025-6029.
[19] Cao YC, Jin R, Mirkin CA: Nanoparticles with Raman spectroscopic fingerprints forDNA and RNA detection. Science2002,297:1536-1540.
[20] Taton TA, Lu G, Mirkin CA: Two-color labeling of oligonucleotide arrays viasize-selective scattering of nanoparticle probes. Journal of the American ChemicalSociety2001,123:5164.
[21] Petryayeva E, Krull UJ: Localized surface plasmon resonance: nanostructures,bioassays and biosensing--a review. Analytica chimica acta2011,706:8-24.
[22] Hu WP, Chen SJ, Huang KT, Hsu JH, Chen WY, Chang GL, Lai KA: A novelultrahigh-resolution surface plasmon resonance biosensor with an Aunanocluster-embedded dielectric film. Biosensors&bioelectronics2004,19:1465-1471.
[23] He L, Musick MD, Nicewarner SR, Salinas FG, Benkovic SJ, Natan MJ, Keating CD:Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection ofDNA hybridization. Journal of the American Chemical Society2000,122:9071-9077.
[24] Hutter E, Pileni M-P: Detection of DNA hybridization by gold nanoparticle enhancedtransmission surface plasmon resonance spectroscopy. The Journal of PhysicalChemistry B2003,107:6497-6499.
[25] Mao X, Yang L, Su XL, Li Y: A nanoparticle amplification based quartz crystalmicrobalance DNA sensor for detection of Escherichia coli O157:H7. Biosensors&bioelectronics2006,21:1178-1185.
[26] Chen SH, Wu VC, Chuang YC, Lin CS: Using oligonucleotide-functionalized Aunanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor.Journal of microbiological methods2008,73:7-17.
[27] Liu T, Tang Ja, Jiang L: Sensitivity enhancement of DNA sensors by nanogold surfacemodification. Biochemical and biophysical research communications2002,295:14-16.
[28] Patolsky F, Ranjit KT, Lichtenstein A, Willner I: Dendritic amplification of DNAanalysis by oligonucleotide-functionalized Au-nanoparticles. Chem Commun2000:1025-1026.
[29] Weizmann Y, Patolsky F, Willner I: Amplified detection of DNA and analysis ofsingle-base mismatches by the catalyzed deposition of gold on Au-nanoparticles.Analyst2001,126:1502-1504.
[30] M ller R, Csaki A, K hler J, Fritzsche W: Electrical classification of theconcentration of bioconjugated metal colloids after surface adsorption and silverenhancement. Langmuir2001,17:5426-5430.
[31] Park S-J, Taton TA, Mirkin CA: Array-based electrical detection of DNA withnanoparticle probes. Science2002,295:1503-1506.
[32] Cai H, Xu C, He P, Fang Y: Colloid Au-enhanced DNA immobilization for theelectrochemical detection of sequence-specific DNA. Journal of electroanalyticalchemistry2001,510:78-85.
[33] Wang J, Xu D, Kawde A-N, Polsky R: Metal nanoparticle-based electrochemicalstripping potentiometric detection of DNA hybridization. Analytical chemistry2001,73:5576-5581.
[2] Tang S, Zhang Q, Yu J, Liu Y, Sha W, Sun H, Fan L, Gu J, Hao X, Yao L: Extensivelydrug-resistant tuberculosis, China. Emerging infectious diseases2011,17:558.
[3] Diedrich CR, Flynn JL: HIV-1/mycobacterium tuberculosis coinfection immunology:how does HIV-1exacerbate tuberculosis? Infect Immun2011,79:1407-1417.
[4] Balabanova Y, Tchernyshev V, Tsigankov I, Maximova S, Mikheeva N, FedyukovitchL, Kuznetsov S, Fedorin I, Drobniewski F: Analysis of undiagnosedtuberculosis-related deaths identified at post-mortem among HIV-infected patients inRussia: a descriptive study. BMC infectious diseases2011,11:276.
[5] Weyer K, Carai S, Nunn P: Viewpoint TB Diagnostics: What Does the World ReallyNeed? The Journal of infectious diseases2011,204Suppl4:S1196-1202.
[6] Morisset D, Stebih D, Cankar K, Zel J, Gruden K: Alternative DNA amplificationmethods to PCR and their application in GMO detection: a review. European FoodResearch and Technology2008,227:1287-1297.
[7] Lee MF, Chen YH, Hsu HJ, Peng CF: One-tube loop-mediated isothermalamplification combined with restriction endonuclease digestion and ELISA forcolorimetric detection of resistance to isoniazid, ethambutol and streptomycin inMycobacterium tuberculosis isolates. Journal of microbiological methods2010,83:53-58.
[8] Zhang D, Wu J, Ye F, Feng T, Lee I, Yin B: Amplification of circularizable probes forthe detection of target nucleic acids and proteins. Clinica chimica acta; internationaljournal of clinical chemistry2006,363:61-70.
[9] Schweitzer B, Kingsmore S: Combining nucleic acid amplification and detection.Curr Opin Biotech2001,12:21–27.
[10] Nallur G, Luo C, Fang L, Cooley S, Dave V, Lambert J, Kukanskis K, Kingsmore S,Lasken R, Schweitzer. B: single amplification by rolling circle amplification on DNAmicroarrays. Nucleic acids research2001,29:e118.
[11] Banér J, Nilsson M, Isaksson A, Mendel-Hartvig M, Antson D, Ulf Landegren U:More keys to padlock probes mechanisms for high-throughput nucleic acid analysis.Curr Opin Biotech2001,12:11–15.
[12] Banér J, Nilsson M, Mendel-Hartvig M, Landegren U: Signal amplification ofpadlock probes by rolling circle replication. Nucleic acids research1998,26:5073-5078.
[13] Demidov VV: Rolling-circle amplification in DNA diagnostic: the power of simplicity.Expert Rev Mol Dtagn2002,2:542-548.
[14] Homola J: Surface Plasmon Resonance Sensors for Detection of Chemical andbiological species. Chem Rev2008,108462-493.
[15] Bailey RC, Qavi AJ, Washburn AL, Byeon JY: Label-free technologies forquantitative multiparameter biological analysis. Analytical and bioanalyticalchemistry2009,394:121-135.
[16] Gusev Y, Sparkowski J, Raghunathan A, Ferguson Jr H, Montano J, Bogdan N,Schweitzer B, Wiltshire S, Kingsmore SF, Maltzman W: Rolling circle amplification:a new approach to increase sensitivity for immunohistochemistry and flow cytometry.The American journal of pathology2001,159:63-69.
[17] Blanco L, Bernad A, Martin G, Garmendia C, Salas M: Highly Efficient DNASynthesis by the Phage Φ29DNA Polymerase. Biological Chemistry1988264:8935-8940.
[18] Liu L, Tang Z, Wang K, Tan W, Li J, Guo Q, Meng X, Ma C: Using molecular beaconto monitor activity of E. coli DNA ligase. Analyst2005,130:350-357.
[19] Tong J, Cao W, Barany F: Biochemical properties of a high fidelity DNA ligase fromThermus species AK16D. Nucleic acids research1999,27:788-794.
[20] Yao C, Zhu T, Tang J, Wu R, Chen Q, Chen M, Zhang B, Huang J, Fu W:Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor.Biosensors&bioelectronics2008,23:879-885.
[21] Xia H, Wang F, Huang Q, Huang J, Chen M, Wang J, Yao C, Chen Q, Cai G, Fu W:Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance NucleicAcid Biosensor Array Using Au Nanoparticle Signal Amplification. Sensors2008,8:6453-6470.
[22] Wang J, Luo Y, Zhang B, Chen M, Huang J, Zhang K, Gao W, Fu W, Jiang T, Liao P:Rapid label-free identification of mixed bacterial infections by surface plasmonresonance. Journal of translational medicine2011,9:85.
[23] Lopez CA, Daaboul GG, Vedula RS, Ozkumur E, Bergstein DA, Geisbert TW,Fawcett HE, Goldberg BB, Connor JH, Unlu MS: Label-free multiplexed virusdetection using spectral reflectance imaging. Biosensors&bioelectronics2011,26:3432-3437.
[24] Kim YS, Yi SY, Kim J, Kim M, Kim CS, Chung BH, Kim JS: Novel application ofsurface plasmon resonance biosensor chips for measurement of advanced glycationend products in serum of Zucker diabetic fatty rats. Biosensors&bioelectronics2009,25:248-252.
[25] Mitchell JS, Lowe TE: Ultrasensitive detection of testosterone using conjugate linkertechnology in a nanoparticle-enhanced surface plasmon resonance biosensor.Biosensors&bioelectronics2009,24:2177-2183.
[26] Milkani E, Morais S, Lambert CR, McGimpsey WG: Detection of oligonucleotidesystematic mismatches with a surface plasmon resonance sensor. Biosensors&bioelectronics2010,25:1217-1220.
[27] Mannelli I, Courtois V, Lecaruyer P, Roger G, Millot MC, Goossens M, Canva M:Surface plasmon resonance imaging (SPRI) system and real-time monitoring of DNAbiochip for human genetic mutation diagnosis of DNA amplified samples. SensorActuat B-Chem2006,119:583-591.
[28] Florschütz K, Schr ter A, Schmieder S, Chen W, Schweizer P, Sonntag F, Danz N,Baronian K, Kunze G:‘Phytochip’–on-chip detection of phytopathogenic RNAviruses by a new surface plasmon resonance platform. Journal of virological methods2013.
[29] Nedelkov D, Nelson RW: Surface plasmon resonance mass spectrometry: recentprogress and outlooks. TRENDS in Biotechnology2003,21:301-305.
[30] Raitman OA, Katz E, Bückmann AF, Willner I: Integration of polyaniline/poly(acrylic acid) films and redox enzymes on electrode supports: an in situelectrochemical/surface plasmon resonance study of the bioelectrocatalyzed oxidationof glucose or lactate in the integrated bioelectrocatalytic systems. Journal of theAmerican Chemical Society2002,124:6487-6496.
[31] Hu WP, Chen SJ, Huang KT, Hsu JH, Chen WY, Chang GL, Lai KA: A novelultrahigh-resolution surface plasmon resonance biosensor with an Aunanocluster-embedded dielectric film. Biosensors&bioelectronics2004,19:1465-1471.
[32] Wood R: XLII. On a remarkable case of uneven distribution of light in a diffractiongrating spectrum. The London, Edinburgh, and Dublin Philosophical Magazine andJournal of Science1902,4:396-402.
[33] Ritchie R: Plasma losses by fast electrons in thin films. Physical Review1957,106:874.
[34] Stern E, Ferrell R: Surface plasma oscillations of a degenerate electron gas. PhysicalReview1960,120:130.
[35] Kretschmann E: The determination of the optical constants of metals by excitation ofsurface plasmons. z Phys1971,241:313-324.
[36] Liedberg B, Nylander C, Lunstr m I: Surface plasmon resonance for gas detectionand biosensing. Sensors and actuators1983,4:299-304.
[37] Dell’Atti D, Tombelli S, Minunni M, Mascini M: Detection of clinically relevantpoint mutations by a novel piezoelectric biosensor. Biosensors and Bioelectronics2006,21:1876-1879.
[38] Chen M, Liu M, Yu L, Cai G, Chen Q, Wu R, Wang F, Zhang B, Jiang T, Fu W:Construction of a novel peptide nucleic acid piezoelectric gene sensor microarraydetection system. Journal of nanoscience and nanotechnology2005,5:1266-1272.
[39] D'Agata R, Corradin R, Ferretti C, Zanoli L, Gatti M, Marchelli R, Spoto G:Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhancedsurface plasmon resonance imaging. Biosensors&bioelectronics2010,25:2095-2100.
[40][Wang J, Zhou HS: Aptamer-based Au nanoparticles-enhanced surface plasmonresonance detection of small molecules. Analytical chemistry2008,80:7174-7178.
[41] Zynio SA, Samoylov AV, Surovtseva ER, Mirsky VM, Shirshov YM: Bimetalliclayers increase sensitivity of affinity sensors based on surface plasmon resonance.Sensors2002,2:62-70.
[42] Hanken DG, Jordan CE, Frey BL, Corn RM: Surface plasmon resonancemeasurements of ultrathin organic films at electrode surfaces. ElectroanalyticalChemistry1998,20:141-226.
[43] Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA: Selective colorimetricdetection of polynucleotides based on the distance-dependent optical properties ofgold nanoparticles. Science1997,277:1078-1081.
[44] Zhang S, Wu Z, Shen G, Yu R: A label-free strategy for SNP detection with highfidelity and sensitivity based on ligation-rolling circle amplification and intercalatingof methylene blue. Biosensors&bioelectronics2009,24:3201-3207.
[45] Yao C, Xiang Y, Deng K, Xia H, Fu W: Sensitive and specific HBV genomic DNAdetection using RCA-based QCM biosensor. Sensor Actuat B-Chem2013.
[46] Lucarelli F, Marrazza G, Turner AP, Mascini M: Carbon and gold electrodes aselectrochemical transducers for DNA hybridisation sensors. Biosensors andBioelectronics2004,19:515-530.
[47] Lai RY, Lagally ET, Lee S-H, Soh H, Plaxco KW, Heeger AJ: Rapid,sequence-specific detection of unpurified PCR amplicons via a reusable,electrochemical sensor. Proceedings of the National Academy of Sciences of theUnited States of America2006,103:4017-4021.
[48] Su Q, Xing D, Zhou X: Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation.Biosensors&bioelectronics2010,25:1615-1621.
[49] Nilsson M, Gullberg M, Dahi F, Szhuai K, Raap AK: Real-time mmonitoring ofrolling-circle amplification using a modificated molecular beacon design. Nucleicacids research2002,30:e66.
[50] McCarthy EL, Bickerstaff LE, da Cunha MP, Millard PJ: Nucleic acid sensing byregenerable surface-associated isothermal rolling circle amplification. Biosensors&bioelectronics2007,22:1236-1244.
[51] Chen Q, Bian Z, Chen M, Hua X, Yao C, Xia H, Kuang H, Zhang X, Huang J, Cai G,Fu W: Real-time monitoring of the strand displacement amplification (SDA) ofhuman cytomegalovirus by a new SDA-piezoelectric DNA sensor system. Biosensors&bioelectronics2009,24:3412-3418.
[52] Wartell RM, Reznikoff WS: Cloning DNA restriction endonuclease fragments withprotruding single-stranded ends. Gene1990,9:307-319.
[53] Dean FB, Nelson JR, Giesler TL, Lasken RS: Rapid amplification of plasmid andphage DNA using phi29DNA polymerase and multiply-primed rolling circleamplification. Genome research2001,11:1095-1099.
[54] Sharma K, Sharma A, Modi M, Singh G, Kaur H, Varma S, Sharma M: PCR detectionof co-infection with Mycobacterium tuberculosis and Mycobacterium avium in AIDSpatients with meningitis. J Med Microbiol2012,61:1789-1791.
[55] Larsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M: In situgenotyping individual DNA molecules by target-primed rolling-circle amplification ofpadlock probes. Nature methods2004,1:227-232.
[56] Andersson C, Henriksson S, Magnusson KE, Nilsson M, Mirazimi A: In situ rollingcircle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV)complementary and viral RNA. Virology2012,426:87-92.
[57] Henriksson S, Blomstrom AL, Fuxler L, Fossum C, Berg M, Nilsson M: Developmentof an in situ assay for simultaneous detection of the genomic and replicative form ofPCV2using padlock probes and rolling circle amplification. Virology journal2011,8:37.
[58] Motre A, Kong R, Li Y: Improving isothermal DNA amplification speed for the rapiddetection of Mycobacterium tuberculosis. Journal of microbiological methods2011,84:343-345.
[59] Torres-Chavolla E, Alocilja EC: Nanoparticle based DNA biosensor for tuberculosisdetection using thermophilic helicase-dependent isothermal amplification. Biosensors&bioelectronics2011,26:4614-4618.
[60] Long Y, Zhou X, Xing D: Sensitive and isothermal electrochemiluminescencegene-sensing of Listeria monocytogenes with hyperbranching rolling circleamplification technology. Biosensors&bioelectronics2011,26:2897-2904.
[61] Aryan E, Makvandi M, Farajzadeh A, Huygen K, Bifani P, Mousavi SL, Fateh A,Jelodar A, Gouya MM, Romano M: A novel and more sensitive loop-mediatedisothermal amplification assay targeting IS6110for detection of Mycobacteriumtuberculosis complex. Microbiological research2010,165:211-220.
[62] Prabhakar N, Arora K, Arya SK, Solanki PR, Iwamoto M, Singh H, Malhotra BD:Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance.Analyst2008,133:1587-1592.
[63] Ramaswamy S, Musser JM: Molecular genetic basis of antimicrobial agent resistancein Mycobacterium tuberculosis:1998update. Tubercle and Lung Disease1998,79:3–29.
[64] Tracevska T, Jansone I, Nodieva A, Marga O, Skenders G, Baumanis V:Characterisation of rpsL, rrs and embB mutations associated withstreptomycin and ethambutol resistance in Mycobacterium tuberculosis.Research in microbiology2004,155:830-834.
[65] Heym B, Alzari PM, Honore N, Cole ST: Missense mutations in the catalase‐peroxidase gene, katG, are associated with isoniazid resistance in Mycobacteriumtuberculosis. Molecular microbiology1995,15:235-245.
[66] Miller LP, Crawford JT, Shinnick TM: The rpoB gene of Mycobacterium tuberculosis.Antimicrobial agents and chemotherapy1994,38:805-811.
[67] Sreevatsan S, Stockbauer KE, Pan X, Kreiswirth BN, Moghazeh SL, Jacobs W,Telenti A, Musser JM: Ethambutol resistance in Mycobacterium tuberculosis: criticalrole of embB mutations. Antimicrobial agents and chemotherapy1997,41:1677-1681.
[68] Finken M, Kirschner P, Meier A, Wrede A, B ttger EC: Molecular basis ofstreptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomalprotein S12gene and point mutations within a functional16S ribosomal RNApseudoknot. Molecular microbiology1993,9:1239-1246.
[69] Haas WH, Schilke K, Brand J, Amthor B, Weyer K, Fourie PB, Bretzel G, Sticht-GrohV, Bremer HJ: Molecular analysis of katG gene mutations in strains ofMycobacterium tuberculosis complex from Africa. Antimicrobial agents andchemotherapy1997,41:1601-1603.
[70] Kiepiela P, Bishop K, Smith A, Roux L, York D: Genomic mutations in the katG, inhAand aphC genes are useful for the prediction of isoniazid resistance in Mycobacteriumtuberculosis isolates from Kwazulu Natal, South Africa. Tubercle and lung disease:the official journal of the International Union against Tuberculosis and Lung Disease2000,80:47.
[71] Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, Collins D,de Lisle G, Jacobs W: inhA, a gene encoding a target for isoniazid and ethionamide inMycobacterium tuberculosis. Science1994,263:227-230.
[72] Mikhailovich V, Lapa S, Gryadunov D, Sobolev A, Strizhkov B, Chernyh N,Skotnikova O, Irtuganova O, Moroz A, Litvinov V: Identification of rifampin-resistant Mycobacterium tuberculosis strains by hybridization, PCR, and ligasedetection reaction on oligonucleotide microchips. Journal of clinical microbiol-ogy2001,39:2531-2540.
[73] Ahmad S, Mokaddas E: The occurrence of rare rpoB mutations inrifampicin-resistant clinical Mycobacterium tuberculosis isolates fromKuwait. International journal of antimicrobial agents2005,26:205-212.
[74] Plinke C, Rüsch-Gerdes S, Niemann S: Significance of mutations in embB codon306for prediction of ethambutol resistance in clinical Mycobacterium tuberculosisisolates. Antimicrobial agents and chemotherapy2006,50:1900-1902.
[75] Dobner P, Bretzel G, Rüsch-Gerdes S, Feldmann K, Rifai M, L scher T, Rinder H:Geographic variation of the predictive values of genomic mutations associated withstreptomycin resistance in Mycobacterium tuberculosis. Molecular andcellular probes1997,11:123-126.
[76] Yao C, Zhu T, Li Y, Zhang L, Zhang B, Huang J, Fu W: Detection of rpoB, katG andinhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqingas determined by microarray Clin Microbiol Infect2010,16:1639–1643.
[77] Telenti A, Imboden P, Marchesi F, Matter L, Schopfer K, Bodmer T, Lowrie D,Colston M, Cole S: Detection of rifampicin-resistance mutations in Mycobacteriumtuberculosis. The Lancet1993,341:647-651.
[78] Caoili JC, Mayorova A, Sikes D, Hickman L, Plikaytis BB, Shinnick TM: Evaluationof the TB-Biochip oligonucleotide microarray system for rapid detection of rifampinresistance in Mycobacterium tuberculosis. Journal of clinical microbiology2006,44:2378-2381.
[79] Brossier F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W: Performance of thegenotype MTBDR line probe assay for detection of resistance to rifampin andisoniazid in strains of Mycobacterium tuberculosis with low-and high-level resistance.Journal of clinical microbiology2006,44:3659-3664.
[80] Duman M, Piskin E: Detection of Mycobacterium tuberculosis complex andMycobacterium gordonae on the same portable surface plasmon resonance sensor.Biosensors&bioelectronics2010,26:908-912.
[81] Marín M, De Viedma DG, Ruíz-Serrano MJ, Bouza E: Rapid direct detection ofmultiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosisin respiratory samples by real-time PCR. Antimicrobial agents and chemotherapy2004,48:4293-4300.
[82] Pai M, Kalantri S, Dheda K: New tools and emerging technologies for the diagnosisof tuberculosis: part II. Active tuberculosis and drug resistance. Expert review ofmolecular diagnostics2006,6:423-432.
[83] Hatch A, Takeshi S, Misasi J, Smith LC: Rolling circle amplification of DNAimmobilized on solid surfaces and its application to multiplex mutation detetion.Genet Anal-Biomole199915:35-40.
[84] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, KingsmoreSF, Lizardi PM, Ward DC: Immunoassays with rolling circle DNA amplification: aversatile platform for ultrasensitive antigen detection. Proceedings of the NationalAcademy of Sciences2000,97:10113-10119.
[85] Thomas DC, Nardone GA, Randall SK: Amplification of padlock probes for DNAdiagnostics by cascade rolling circle amplification or the polymerase chain reaction.Archives of pathology&laboratory medicine1999,123:1170-1176.
[86] Wilkomson D: Third wave technologies' invader assays for nucleic acid detection.The Scientist1993,13:16-18.
[87] Hardenbol p, Baner J, Jain M, Nilsson M, Namsaraev E: Multiplexed genotyping withsequence-tagged molecular inversion probes. Nature biotechnology2003,21:673-678.
[88] Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Kwiatkowski RW,Sander TJ, de Arruda M, Arco DA: Polymorphism identification and quantitativedetection of genomic DNA by invasive cleavage of oligonucleotide probes. Naturebiotechnology1999,17:292-296.
[89] ALLAWI HT, DAHLBERG JE, OLSON S, LUND E, OLSON M, MA W-P, TAKOVAT, NERI BP, LYAMICHEV VI: Quantitation of microRNAs using a modified Invaderassay. Rna2004,10:1153-1161.
[90] Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C-a, Horn T, Ahle D, DetmerJ, Shen L-P: A branched DNA signal amplification assay for quantification of nucleicacid targets below100molecules/ml. Nucleic acids research1997,25:2979-2984.
[91] Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS,Perrillo RP: Quantitation of HBV DNA in human serum using a branched DNA(bDNA) signal amplification assay. American journal of clinical pathology1995,104:537.
[92] Pachl C, Todd JA, Kern DG, Sheridan PJ, Fong S-J, Stempien M, Hoo B, Besemer D,Yeghiazarian T, Irvine B: Rapid and precise quantification of HIV-1RNA in plasmausing a branched DNA signal amplification assay. JAIDS Journal of AcquiredImmune Deficiency Syndromes1995,8:446&hyhen.
[93] Zhang DY, Winfree E: Robustness and modularity properties of a non-covalent DNAcatalytic reaction. Nucleic acids research2010,38:4182-4197.
[94] Choi HM, Chang JY, Trinh LA, Padilla JE, Fraser SE, Pierce NA: Programmable insitu amplification for multiplexed imaging of mRNA expression. Naturebiotechnology2010,28:1208-1212.
[95] Huang J, Wu Y, Chen Y, Zhu Z, Yang X, Yang CJ, Wang K, Tan W: Pyrene‐ExcimerProbes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acidsin Complex Biological Fluids. Angewandte Chemie International Edition2011,50:401-404.
[96] Zhang B, Liu B, Tang D, Niessner R, Chen G, Knopp D: DNA-Based HybridizationChain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection ofProteins. Analytical chemistry2012,84:5392-5399.
[97] Wang C, Zhou H, Zhu W, Li H, Jiang J, Shen G, Yu R: Ultrasensitive electrochemicalDNA detection based on dual amplification of circular strand-displacementpolymerase reaction and hybridization chain reaction. Biosensors and Bioelectronics2013.
[98] Dirks RM, Pierce NA: Triggered amplification by hybridization chain reaction.Proceedings of the National Academy of Sciences of the United States of America2004,101:15275-15278.
[99] Reichert J, Csáki A, K hler JM, Fritzsche W: Chip-based optical detection of DNAhybridization by means of nanobead labeling. Analytical chemistry2000,72:6025-6029.
[100] Cao YC, Jin R, Mirkin CA: Nanoparticles with Raman spectroscopic fingerprints forDNA and RNA detection. Science2002,297:1536-1540.
[101] Taton TA, Lu G, Mirkin CA: Two-color labeling of oligonucleotide arrays viasize-selective scattering of nanoparticle probes. Journal of the American ChemicalSociety2001,123:5164.
[102] Petryayeva E, Krull UJ: Localized surface plasmon resonance: nanostructures,bioassays and biosensing--a review. Analytica chimica acta2011,706:8-24.
[103] He L, Musick MD, Nicewarner SR, Salinas FG, Benkovic SJ, Natan MJ, Keating CD:Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection ofDNA hybridization. Journal of the American Chemical Society2000,122:9071-9077.
[104] Hutter E, Pileni M-P: Detection of DNA hybridization by gold nanoparticle enhancedtransmission surface plasmon resonance spectroscopy. The Journal of PhysicalChemistry B2003,107:6497-6499.
[105] Mao X, Yang L, Su XL, Li Y: A nanoparticle amplification based quartz crystalmicrobalance DNA sensor for detection of Escherichia coli O157:H7. Biosensors&bioelectronics2006,21:1178-1185.
[106] Chen SH, Wu VC, Chuang YC, Lin CS: Using oligonucleotide-functionalized Aunanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor.Journal of microbiological methods2008,73:7-17.
[107] Liu T, Tang Ja, Jiang L: Sensitivity enhancement of DNA sensors by nanogold surfacemodification. Biochemical and biophysical research communications2002,295:14-16.
[108] Patolsky F, Ranjit KT, Lichtenstein A, Willner I: Dendritic amplification of DNAanalysis by oligonucleotide-functionalized Au-nanoparticles. Chem Commun2000:1025-1026.
[109] Weizmann Y, Patolsky F, Willner I: Amplified detection of DNA and analysis ofsingle-base mismatches by the catalyzed deposition of gold on Au-nanoparticles.Analyst2001,126:1502-1504.
[110] M ller R, Csaki A, K hler J, Fritzsche W: Electrical classification of theconcentration of bioconjugated metal colloids after surface adsorption and silverenhancement. Langmuir2001,17:5426-5430.
[111] Park S-J, Taton TA, Mirkin CA: Array-based electrical detection of DNA withnanoparticle probes. Science2002,295:1503-1506.
[112] Cai H, Xu C, He P, Fang Y: Colloid Au-enhanced DNA immobilization for theelectrochemical detection of sequence-specific DNA. Journal of electroanalyticalchemistry2001,510:78-85.
[113] Wang J, Xu D, Kawde A-N, Polsky R: Metal nanoparticle-based electrochemicalstripping potentiometric detection of DNA hybridization. Analytical chemistry2001,73:5576-5581.
[114] Teresa. Torre, Mattias. Stromberg, Camilla. Russell, Jenny. Goransson, Mats. Nilsson,Peter. Svedlindh, Maria. Stromme: Investigation of Immobilization of FunctionalizedMagnetic Nanobeads in Rolling Circle Amplified DNA Coils. J. Phys. Chem. B2010,114:3707-3713.
[1] Hatch A, Takeshi S, Misasi J, Smith LC: Rolling circle amplification of DNAimmobilized on solid surfaces and its application to multiplex mutation detetion.Genet Anal-Biomole199915:35-40.
[2] Banér J, Nilsson M, Isaksson A, Mendel-Hartvig M, Antson D, Ulf Landegren U:More keys to padlock probes mechanisms for high-throughput nucleic acid analysis.Curr Opin Biotech2001,12:11–15.
[3] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, KingsmoreSF, Lizardi PM, Ward DC: Immunoassays with rolling circle DNA amplification: aversatile platform for ultrasensitive antigen detection. Proceedings of the NationalAcademy of Sciences2000,97:10113-10119.
[4] Thomas DC, Nardone GA, Randall SK: Amplification of padlock probes for DNAdiagnostics by cascade rolling circle amplification or the polymerase chain reaction.Archives of pathology&laboratory medicine1999,123:1170-1176.
[5] Wilkomson D: Third wave technologies' invader assays for nucleic acid detection.The Scientist1993,13:16-18.
[6] Hardenbol p, Baner J, Jain M, Nilsson M, Namsaraev E: Multiplexed genotyping withsequence-tagged molecular inversion probes. Nature biotechnology2003,21:673-678.
[7] Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Kwiatkowski RW,Sander TJ, de Arruda M, Arco DA: Polymorphism identification and quantitativedetection of genomic DNA by invasive cleavage of oligonucleotide probes. Naturebiotechnology1999,17:292-296.
[8] ALLAWI HT, DAHLBERG JE, OLSON S, LUND E, OLSON M, MA W-P, TAKOVAT, NERI BP, LYAMICHEV VI: Quantitation of microRNAs using a modified Invaderassay. Rna2004,10:1153-1161.
[9] Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C-a, Horn T, Ahle D, DetmerJ, Shen L-P: A branched DNA signal amplification assay for quantification of nucleicacid targets below100molecules/ml. Nucleic acids research1997,25:2979-2984.
[10] Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS,Perrillo RP: Quantitation of HBV DNA in human serum using a branched DNA(bDNA) signal amplification assay. American journal of clinical pathology1995,104:537.
[11] Pachl C, Todd JA, Kern DG, Sheridan PJ, Fong S-J, Stempien M, Hoo B, Besemer D,Yeghiazarian T, Irvine B: Rapid and precise quantification of HIV-1RNA in plasmausing a branched DNA signal amplification assay. JAIDS Journal of AcquiredImmune Deficiency Syndromes1995,8:446&hyhen.
[12] Zhang DY, Winfree E: Robustness and modularity properties of a non-covalent DNAcatalytic reaction. Nucleic acids research2010,38:4182-4197.
[13] Choi HM, Chang JY, Trinh LA, Padilla JE, Fraser SE, Pierce NA: Programmable insitu amplification for multiplexed imaging of mRNA expression. Nature biotechn-ology2010,28:1208-1212.
[14] Huang J, Wu Y, Chen Y, Zhu Z, Yang X, Yang CJ, Wang K, Tan W: Pyrene‐ExcimerProbes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acidsin Complex Biological Fluids. Angewandte Chemie International Edition2011,50:401-404.
[15] Zhang B, Liu B, Tang D, Niessner R, Chen G, Knopp D: DNA-Based HybridizationChain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection ofProteins. Analytical chemistry2012,84:5392-5399.
[16] Wang C, Zhou H, Zhu W, Li H, Jiang J, Shen G, Yu R: Ultrasensitive electrochemicalDNA detection based on dual amplification of circular strand-displacementpolymerase reaction and hybridization chain reaction. Biosensors and Bioelectronics2013.
[17] Dirks RM, Pierce NA: Triggered amplification by hybridization chain reaction.Proceedings of the National Academy of Sciences of the United States of America2004,101:15275-15278.
[18] Reichert J, Csáki A, K hler JM, Fritzsche W: Chip-based optical detection of DNAhybridization by means of nanobead labeling. Analytical chemistry2000,72:6025-6029.
[19] Cao YC, Jin R, Mirkin CA: Nanoparticles with Raman spectroscopic fingerprints forDNA and RNA detection. Science2002,297:1536-1540.
[20] Taton TA, Lu G, Mirkin CA: Two-color labeling of oligonucleotide arrays viasize-selective scattering of nanoparticle probes. Journal of the American ChemicalSociety2001,123:5164.
[21] Petryayeva E, Krull UJ: Localized surface plasmon resonance: nanostructures,bioassays and biosensing--a review. Analytica chimica acta2011,706:8-24.
[22] Hu WP, Chen SJ, Huang KT, Hsu JH, Chen WY, Chang GL, Lai KA: A novelultrahigh-resolution surface plasmon resonance biosensor with an Aunanocluster-embedded dielectric film. Biosensors&bioelectronics2004,19:1465-1471.
[23] He L, Musick MD, Nicewarner SR, Salinas FG, Benkovic SJ, Natan MJ, Keating CD:Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection ofDNA hybridization. Journal of the American Chemical Society2000,122:9071-9077.
[24] Hutter E, Pileni M-P: Detection of DNA hybridization by gold nanoparticle enhancedtransmission surface plasmon resonance spectroscopy. The Journal of PhysicalChemistry B2003,107:6497-6499.
[25] Mao X, Yang L, Su XL, Li Y: A nanoparticle amplification based quartz crystalmicrobalance DNA sensor for detection of Escherichia coli O157:H7. Biosensors&bioelectronics2006,21:1178-1185.
[26] Chen SH, Wu VC, Chuang YC, Lin CS: Using oligonucleotide-functionalized Aunanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor.Journal of microbiological methods2008,73:7-17.
[27] Liu T, Tang Ja, Jiang L: Sensitivity enhancement of DNA sensors by nanogold surfacemodification. Biochemical and biophysical research communications2002,295:14-16.
[28] Patolsky F, Ranjit KT, Lichtenstein A, Willner I: Dendritic amplification of DNAanalysis by oligonucleotide-functionalized Au-nanoparticles. Chem Commun2000:1025-1026.
[29] Weizmann Y, Patolsky F, Willner I: Amplified detection of DNA and analysis ofsingle-base mismatches by the catalyzed deposition of gold on Au-nanoparticles.Analyst2001,126:1502-1504.
[30] M ller R, Csaki A, K hler J, Fritzsche W: Electrical classification of theconcentration of bioconjugated metal colloids after surface adsorption and silverenhancement. Langmuir2001,17:5426-5430.
[31] Park S-J, Taton TA, Mirkin CA: Array-based electrical detection of DNA withnanoparticle probes. Science2002,295:1503-1506.
[32] Cai H, Xu C, He P, Fang Y: Colloid Au-enhanced DNA immobilization for theelectrochemical detection of sequence-specific DNA. Journal of electroanalyticalchemistry2001,510:78-85.
[33] Wang J, Xu D, Kawde A-N, Polsky R: Metal nanoparticle-based electrochemicalstripping potentiometric detection of DNA hybridization. Analytical chemistry2001,73:5576-5581.