Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay

Magnetic nanoparticles (MNPs) have been receiving increasing attention because of its great potentials in bioseparation. However, the separation products are difficult to be detected by general method due to their extremely small size. Here, we demonstrate that MNPs can greatly enhance the signal of surface plasmon resonance spectroscopy (SPR). Features of MNPs-aptamer conjugates as a powerful amplification reagent for ultrasensitive immunoassay are reported in this work for the first time. In order to evaluate the sensing ability of MNPs-aptamer conjugates as an amplification reagent, a sandwich SPR sensor is constructed by using thrombin as model analyte. Thrombin, captured by immobilized anti-thrombin aptamer on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 0.017 nM after MNPs-aptamer conjugates is used as amplification reagent. At the same time, the excellent selectivity of the present biosensor is also confirmed by using three kinds of proteins (BSA, human IgM and human IgE) as controls. These results confirm that MNPs is a powerful sandwich element and an excellent amplification reagent for SPR based sandwich immunoassay and SPR has a great potential for the detection of MNPs-based bioseparation products.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

Multianalyte Electrochemical Biosensor Based on Aptamer‐ and Nanoparticle‐Integrated Bio‐Barcode Amplification

In the present work, a signal‐on electrochemical sensing strategy for the simultaneous detection of adenosine and thrombin is developed based on switching structures of aptamers. An Au electrode as the sensing surface is modified with two kinds of thiolated capture probes complementary to the linker DNA that contains either an adenosine aptamer or thrombin aptamer. The capture probes hybridize with their corresponding linker DNA, which has prehybridized with the reporter DNA loaded onto the gold nanoparticles (AuNPs). The AuNP contained two kinds of bio‐barcode DNA: one is complementary to the linker DNA (reporter), whereas the other is not (signal) and is tagged with different metal sulfide nanoparticles. Thus a “sandwich‐type” sensing interface is fabricated for adenosine and thrombin. With the introduction of adenosine and thrombin, the aptamer parts bind with their targets and fold to form the complex structures. As a result, the bio‐barcoded AuNPs are released into solution. The metal sulfide nanoparticles are measured by anodic stripping voltammetry (ASV), and the concentrations of adenosine and thrombin are proportional to the signal of either metal ion. With the dual amplification of the bio‐barcoded AuNP and the preconcentration of metal ions through ASV technology, detection limits as low as 6.6×10^?12 M for adenosine and 1.0×10^?12 M for thrombin are achieved. The sensor exhibits excellent selectivity and detectability in biological samples.     

Determination of Lysozyme by Thiol-Terminated Aptamer-Based Surface Plasmon Resonance

A sensitive and selective surface plasmon resonance (SPR) aptasensor has been developed for the real-time determination of lysozyme. The thiol-terminated lysozyme aptamer was covalently attached to the surface of sensor through Au–S bonding. In the presence of lysozyme, the aptamer captured lysozyme on the surface and enhanced the SPR signal that was proportional to the concentration of lysozyme. Under the optimized conditions, the SPR signal was linear with lysozyme concentration from 1 to 100?µmol/L. The detection limit for lysozyme was 0.5?µmol/L. The aptasensor also provided high specificity for lysozyme and was unaffected by other proteins. This aptasensor provides rapid, simple, and sensitive determination of lysozyme detection and a promising strategy for other proteins.     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

     

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H₂O₂-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.     

基于计时库仑技术的可再生型三磷酸腺苷适配体电化学传感器的研究

构建了一种可再生型三磷酸腺苷(ATP)适配体计时库仑电化学传感器.将一条短链DNA通过AuS键自组装固定在电极表面, ATP的核酸适配体与该短链DNA杂交而结合在电极表面.带负电的DNA通过静电吸引结合电解液中的六氨合钌(RuHex)阳离子.当传感器和靶分子ATP孵育后,ATP与核酸适配体结合,使适配体链从电极表面解离,电极表面吸附的DNA量减少,结合RuHex的量随之降低.通过计时库仑技术检测RuHex响应信号降低的量 ,可以对ATP进行定量测定.此传感器的电化学响应信号与ATP浓度对数值呈线性关系,线性检测范围为0.001~100 μmol/L,检出限(S/N=3)为0.5 nmol/L.此传感器检测靶分子ATP后,可以通过简单的操作步骤再生,再生5次后的响应信号为初始信号的90%以上.采用此传感器检测大鼠脑透析液中ATP的含量为(19.2±3.7) nmol/L (n=3).     

A surface plasmon resonance sensor on a compact disk-type microfluidic device

A surface plasmon resonance (SPR) sensor on a compact disk (CD)-type microfluidic device was developed to miniaturize the elements of a complete analytical system, pump and valves. The CD-type microfluidic device was fabricated by attaching a polydimethylsiloxane disk plate that contained microchannels and reservoirs to a flat polycarbonate disk plate that contained grating films with a thin layer of Au. The optical system of the SPR sensor and the theory for its operation are based on the principle of a grating coupled-type SPR. The sample and reagent solutions in the reservoirs on the CD-type microfluidic device were sequentially introduced into the detection chamber by centrifugal force generated by the rotation of the microfluidic device. The variation of resonance wavelength was dependent on the refractive index of the sample solution. This CD-type SPR sensor was successfully used in an immunoassay of immunoglobulin A (IgA). The anti-IgA, blocking reagent, sample and washing solution in the reservoirs were sequentially introduced into the detection chamber by changing the frequency of rotation of the microfluidic device. IgA in the sample solution was adsorbed to the anti-IgA immobilized on the Au thin layer in the detection chamber and was then detected by the SPR sensor.     

Amplified surface plasmon resonance based DNA biosensors, aptasensors, and Hg2+ sensors using hemin/G-quadruplexes and Au nanoparticles

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.     

Gold nanoparticle-enhanced surface plasmon resonance measurement with a highly sensitive quantification for human tissue inhibitor of metalloproteinases-2

Two different methods for the quantification of human tissue inhibitor of metalloproteinases-2 (TIMP-2) were developed using surface plasmon resonance (SPR) and gold nanoparticles for signal enhancement. The first method, a competitive assay, used TIMP-2 immobilized to the sensor surface and the inactive form of matrix metalloproteinase-2 (proMMP-2) (EC 3.4.24.24) adsorbed to gold nanoparticles. The sensor signals resulting from the interaction of MMP-2-gold nanoparticles with immobilized TIMP-2 were inversely proportional to the amounts of TIMP-2 of the sample. The measuring range for TIMP-2 was about 15–180 pM. The second method, a one-step sandwich assay, used proMMP-2 immobilized to the sensor surface and an anti-TIMP-2 monoclonal antibody coupled to gold nanoparticles. The lower detection limit of this assay format was 0.5 pM of TIMP-2. The binding signals were highly reproducible up to 100 pM of the inhibitor. The improvements obtained in TIMP-2 quantification over already existing tests could contribute to a better understanding and diagnosis of diseases like cancer.     

Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance

Cysteine modified NH(2)-end peptide nucleic acid (PNA) (24-mer) probe and 5'-thiol end labeled deoxyribonucleic acid (DNA) probes specific to Mycobacterium tuberculosis have been immobilized onto BK-7 gold coated glass plates for the detection of complementary, one-base mismatch, non-complementary targets and complementary target sequence in genomic DNA of Mycobacterium tuberculosis using a surface plasmon resonance (SPR) technique. The DNA/Au and PNA/Au bio-electrodes have been characterized using contact angle, atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) techniques, respectively. It is revealed that there is a 252 millidegrees SPR angle change in the case of PNA immobilization and 205 millidegrees for DNA immobilization, indicating increased amount of immobilized PNA molecules. Hybridization studies reveal that there is no binding of the non-complementary target to DNA/Au and PNA/Au electrode. Compared to the DNA/Au bioelectrode, PNA/Au electrode has been found to be more efficient for detection of one-base mismatch sequence. The PNA/Au bioelectrode shows better detection limit (1.0 ng ml(-1)) over the DNA-Au bioelectrode (3.0 ng ml(-1)). The values of the association (k(a)) and dissociation rate constant (k(d)) for the complementary sequence in case of the PNA/Au bioelectrode have been estimated as 8.5 x 10(4) m(-1) s(-1) and 3.6 x 10(-3) s(-1), respectively.     

基于多边形金纳米颗粒的非标记型可卡因适体传感器的研制

实验合成了多边形金纳米颗粒,通过壳聚糖(CHIT)将合成的多边形金纳米颗粒固定在玻碳电极表面,然后通过自组装技术将带巯基的捕获DNA探针固定在修饰有多边形金纳米颗粒的电极表面,利用杂交反应使可卡因适体与DNA捕获探针结合,制成非标记型可卡因适体传感器。以六氨合钌作为电化学指示剂,通过测量传感器与目标物可卡因结合前后电流变化情况对可卡因进行测定。考察了缓冲溶液的pH、可卡因培育时间、扫描速度等对测定的影响。结果表明,在pH为7.40时该传感器的检测范围为1.0×10-10～1.0×10-3 mol/L,检测限为3.0×10-11 mol/L。该传感器制作简单,响应好,抗干扰能力强。     

SPR sensing of bisphenol A using molecularly imprinted nanoparticles immobilized on slab optical waveguide with consecutive parallel Au and Ag deposition bands coexistent with bisphenol A-immobilized Au nanoparticles

A slab-type optical waveguide (s_OWG)-based microfluidic SPR measurement system for bisphenol A was developed. This s_OWG possesses consecutive parallel gold and silver deposition bands in the line of plasmon flow, allowing two individual SPR signals to be independently obtained as a result of the difference in resonant reflection spectra of these metals. As a molecular recognition element, molecularly imprinted polymer nanoparticles (MIP-Np) were employed and immobilized on the surface of each of the gold and silver deposition bands. The resonant reflection spectra were measured on the MIP-Np-immobilized consecutive parallel gold and silver deposition bands coexistent with BPA-AuNp. The Ag-based SPR spectra showed a red shift (0.7 nm) when free BPA (0.1 mM) was passed over the BPA-AuNp/immobilized MIP-Np complexes formed on the s_OWG, unlike the case for the Au deposition band, while a large excess of BPA induced a blue shift due to the competitive desorption of BPA-AuNp from the immobilized MIP-Np on the s_OWG. By using the proposed detection system, binding events of other small molecules could be monitored in conjunction with the use of MIP-Np and labeled-AuNp.     

Label-free electrochemical detection of nanomolar adenosine based on target-induced aptamer displacement

In this work, a label-free electrochemical sensor based on target-induced displacement is reported with adenosine as the model analyte. The sensing substrate is prepared using a gold electrode modified with a self-assembled monolayer of 1,6-hexanedithiol that mediates the assembly of a gold nanoparticle film, which can increase the surface loading of capture probe and enhance the signal. An aptamer for adenosine is applied to hybridizing with the capture probe, yielding a double-stranded complex of the aptamer and the capture probe on the surface. The interaction of adenosine with the aptamer displaces the aptamer sequence and causes it to dissociate from the interface. This results in a decrease in the amount of aptamer/capture probe duplex form, and, accordingly, the desorption of methylene blue, an electroactive indicator bound to the duplex, from the electrode. Then, the redox current of the indicator can reflect the concentration of the analyte. The fabricated sensor is shown to exhibit high sensitivity, desirable selectivity and a three-decade wide linear range.     

Surface plasmon resonance-based trace detection of small molecules by competitive and signal enhancement immunoreaction

A surface plasmon resonance (SPR)-immunosensor for detection of the low molecular weight compound 2,4-dinitorophenol (DNP) at ultra-low concentration has been developed. The sensor strategy is based on a competitive immunoreaction between DNP and a DNP-protein conjugate, namely DNP-bovine serum albumin conjugate (DNP-BSA). Anti-DNP monoclonal antibody was immobilized on a gold thin-film coated SPR-sensor chip by means of a chemical coupling process. DNP-BSA, on contact with the anti-DNP antibody immobilized SPR-immunosensor chip causes an increase in the resonance angle of the sensor chip. The optimum concentration of immobilized antibody on the SPR-sensor chip is 100 μg mL⁻¹. The SPR-immunosensor response for free DNP determination using the competitive immunoreaction had a response time of ca. 15 min. Using this method, DNP could be determined in the concentration range 1 ppt to 1 ppb. The SPR signal for ppt levels of DNP was enhanced by a factor of three by subsequently treating immuno-bound DNP-BSA with a secondary anti-DNP antibody.     

非标记夹心式电化学可卡因适体传感器的研究

设计一种基于双链核酸适体的非标记夹心式电化学适体传感器, 建立简单、高灵敏度的可卡因分析方法. 首先将末端巯基修饰的捕获适体探针组装在金电极表面, 构建可卡因适体传感器. 该传感器与目标分子可卡因和部分互补的检测适体探针作用后, 在电极表面形成适体/可卡因/适体复合物. 以六氨合钌为信号分子, 基于单链适体和适体/可卡因/适体复合物对六氨合钌吸附量的不同, 通过计时电量法检测电极表面吸附六氨合钌的还原电量, 进行可卡因的分析检测. 在优化的条件下, 还原电量与可卡因浓度在1～50 mmol/L范围内呈良好的线性关系, 检出限为0.1 mmol/L. 用于血清中可卡因的检测, 回收率为96.4%～104%. 该方法简单, 灵敏度高, 可作为一种通用型的适体传感器模型.     

Femtomolar electrochemical detection of DNA targets using metal sulfide nanoparticles

A new electrochemical DNA sensor providing detection capabilities down to 100 attomol of target DNA has been developed. The method applies CdS, ZnS, and PbS nanoparticles conjugated with short DNA sequences which are immobilized via hybridization with complementary sequences on a gold surface. When the DNA target is added, it can be identified by ousting the existing hybridization between one of the DNA-nanoparticle conjugates and the surface DNA. The nanoparticles remaining at the surface are detected by stripping voltammetry. The setup is constructed to give a signal-off response with a build-in control signal as only one of two different metal sulfide signaling probes on the surface is removed by hybridization with the DNA target. The competition assay is, in principle, label-free since no labels are required for detection after addition of DNA target. The dissociation of PbS nanoparticles from the surface after addition of the DNA target has been imaged by fluid phase AFM.     

核酸修饰的金纳米粒子用于分光光度法检测卡那霉素

建立了一种基于核酸修饰的金纳米粒子(Au NPs)检测卡那霉素的方法。该方法利用卡那霉素与适配体的特异性结合,游离适配体的部分互补序列,诱导核酸修饰的Au NPs聚集。通过对实验条件进行优化,结果表明在25℃条件下,适配体与其部分互补序列杂交摩尔比为1:1,与目标卡那霉素的作用时间1 h,加入核酸修饰的Au NPs反应2 h时,该方法的线性检测范围为6.3~43.8 nmol/L,检测限为5.3 nmol/L。将该方法应用于牛奶样品中卡那霉素的检测,回收率在95.1%~104.6%之间。     

Label-free aptamer-based chemiluminescence detection of adenosine

We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10⁻⁷ to 1 × 10⁻⁵ M. With a low detection limit of 8 × 10⁻⁸ M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.     

基于微悬臂梁生物传感器检测三磷酸腺苷

基于适配子构建了无标记检测三磷酸腺苷(ATP)的微悬臂梁生物传感器。 将ATP适配子修饰在微悬臂梁阵列中的传感悬臂镀金面上,用来识别ATP,而参比悬臂修饰巯基己醇(MCH)防止非特异性吸附。 ATP与其适配子发生特异性相互作用,使悬臂的上下两个表面产生应力差,导致传感悬臂产生偏转,扣除参比悬臂偏转后其偏转值与ATP的浓度在0.5~5 mmol/L范围内有良好的线性关系,相关系数为0.998,最低检出限为0.06 mmol/L。 该微悬臂梁生物传感器响应快速、操作简单,并且对ATP具有良好的特异性。