Portable and quantitative detection of protein biomarkers and small molecular toxins using antibodies and ubiquitous personal glucose meters

Developing portable and low-cost methods for quantitative detection of large protein biomarkers and small molecular toxins can play a significant role in controlling and preventing diseases or toxins outbreaks. Despite years of research, most current methods still require laboratory-based or customized devices that are not widely available to the general public for quantitative analysis. We have previously demonstrated the use of personal glucose meters (PGMs) and functional DNAs for the detection of many nonglucose targets. However, the range of targets detectable by functional DNAs is limited at the current stage. To expand the range of targets that can be detected by PGMs, we report here the use of antibodies in combination with sandwich and competitive assays for quantitative detection of protein biomarkers (PSA, with a detection limit of 0.4 ng/mL) and small molecular toxins (Ochratoxin A, with a detection limit of 6.8 ng/mL), respectively. In both assay methods, with invertase conjugates as the link, quantitative detection is achieved via the dependence between the concentrations of the targets in the sample and the glucose measured by PGMs. Given the wide availability of antibodies for numerous targets, the methods demonstrated here can expand the range of target detection by PGMs significantly.     

Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip

We describe a disposable electrochemical immunosensor diagnosis device that integrates the immunochromatographic strip technique with an electrochemical immunoassay and exploits quantum dot (QD, CdS@ZnS) as labels for amplifying signal output. The device takes advantage of the speed and low cost of the conventional immunochromatographic strip test and the high sensitivity of the nanoparticle-based electrochemical immunoassay. A sandwich immunoreaction was performed on the immunochromatographic strip, and the captured QD labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable screen-printed electrode, which is embedded underneath the membrane on the test zone. The new device coupled with a portable electrochemical analyzer shows great promise for in-field and point-of-care quantitative testing of disease-related protein biomarkers. The parameters (e.g., voltammetric measurement of QD labels, antibody immobilization, the loading amount of QD-antibody, and the immunoreaction time) that govern the sensitivity and reproducibility of the device were optimized with IgG model analyte. The voltammetric response of the optimized device is highly linear over the range of 0.1-10 ng mL(-1) IgG, and the limit of detection is estimated to be 30 pg mL(-1) in association with a 7-min immunoreaction time. The detection limit was improved to 10 pg mL(-1) using a 20-min immunoreaction time. The device has been successfully applied for the detection of prostate-specific antigen (PSA) in human serum sample with a detection limit of 20 pg mL(-1). The results were validated by using the commercial PSA enzyme-linked immunosorbent assay kit and showed high consistency. The new disposable electrochemical diagnosis device thus provides a rapid, clinically accurate, and quantitative tool for protein biomarker detection.     

Multiplexed cancer biomarker detection using quartz-based photonic crystal surfaces

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20-99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1-41 pg/mL), resulting in a three-log range of quantitative detection.     

Label-free protein recognition two-dimensional array using nanomechanical sensors

We demonstrate two-dimensional multiplexed real-time, label-free antibody-antigen binding assays by optically detecting nanoscale motions of two-dimensional arrays of microcantilever beams. Prostate specific antigen (PSA) was assayed using antibodies covalently bound to one surface of the cantilevers by two different surface chemistries, while the nonreaction surfaces were passivated by poly(ethylene glycol)-silane. PSA as low as 1 ng/mL was detected while 2 mg/microl of bovine serum albumin induced only negligible deflection on the cantilevers.     

Amplified electrochemical detection of a cancer biomarker by enhanced precipitation using horseradish peroxidase attached on carbon nanotubes

An electrochemical nanoimmunosensor based on multiwall carbon nanotubes (MWCNTs)/gold nanoparticles (AuNPs) was developed for the amplified detection of prostate specific antigen (PSA). The amplified detection was achieved by the enhanced precipitation of 4-chloro-1-naphthol (CN) using a higher number of horseradish peroxidase (HRP) molecules attached on MWCNTs. The PSA nanoimmunosensor was fabricated by immobilizing a monoclonal anti-PSA antibody (anti-PSA) on the AuNP-attached thiolated MWCNT on a gold electrode. The sensor surface was characterized using scanning electron microscope, transmission electron microscope, quartz crystal microbalance, and electrochemical techniques. Cyclic and square wave voltammetric techniques were used to monitor the enhanced precipitation of CN that accumulated on the electrode surface and subsequent decrement in the electrode surface area by monitoring the reduction process of the Fe(CN)(6)(3-)/Fe(CN)(6)(4-) redox couple. Under the optimized experimental condition, the linear range and the detection limit of PSA immunosensor were determined to be 1.0 pg/mL to 10.0 ng/mL and 0.40 ± 0.03 pg/mL, respectively. The validity of the proposed method was compared with an enzyme-linked immunosorbent assay method in various PSA spiked human serum samples.     

Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab(2)). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab(1)) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab(2)) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL(-1) to 8 ng mL(-1). The detection limit of PSA was 8 pg mL(-1) (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.     

Signal enhancement in antibody microarrays using quantum dots nanocrystals: application to potential Alzheimer's disease biomarker screening

The performance of cadmium-selenide/zinc-sulfide (CdSe@ZnS) quantum dots (QDs) and the fluorescent dye Alexa 647 as reporter in an assay designed to detect apolipoprotein E (ApoE) has been compared. The assay is a sandwich immunocomplex microarray that functions via excitation by visible light. ApoE was chosen for its potential as a biomarker for Alzheimer's disease. The two versions of the microarray (QD or Alexa 647) were assessed under the same experimental conditions and then compared to a conventional enzyme-linked immunosorbent assay (ELISA) targeting ApoE. The QDs proved to be highly effective reporters in the microarrays, although their performance strongly varied in function of the excitation wavelength. At 633 nm, the QD microarray gave a limit of detection (LOD) of ～247 pg mL(-1); however, at an excitation wavelength of 532 nm, it provided a LOD of ～62 pg mL(-1), five times more sensitive than that of the Alexa microarray (～307 pg mL(-1)) and seven times more than that of the ELISA (～470 pg mL(-1)). Finally, serial dilutions from a human serum sample were assayed with high sensitivity and acceptable precision and accuracy.     

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.     

Channel and substrate zone two-dimensional resolution for chemiluminescent multiplex immunoassay

A two-dimensional resolution system of channels and substrate zones was proposed for multiplex immunoassay performed with a designed multichannel chemiluminescent (CL) detection device coupled with a single photomultiplier. Using carcinoma antigen 125 (CA 125), carcinoma antigen 153 (CA 153), carcinoma antigen 199 (CA 199), and carcinoembryonic antigen (CEA) as two couples of model analytes, two couples of capture antibodies were immobilized in two channels, respectively. With a sandwich format, the CL substrates for alkaline phosphatase and horseradish peroxidase were delivered into the channels sequentially to perform a multiplex immunoassay after the sample and tracer antibodies were introduced into the channels for on-line incubation. CA 125, CA 153, CA 199, and CEA could be assayed in the ranges of 0.50-80, 2.0-100, and 5.0-150 U/mL and 1.0-70 ng/mL with limits of detection of 0.15, 0.80, and 2.0 U/mL and 0.65 ng/mL at 3sigma, respectively. The whole assay process including regeneration of the device could be completed in 37 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for a simple, automated, and near-simultaneous multianalyte immunoassay.     

利用微型生物光学传感器的竞争免疫检测法检测牛奶中氨苄青霉素

利用微型表面等离子体共振的生物传感器测量了残留在牛奶中的氨苄青霉素的浓度.采用竞争抑制试验的方法,即先将定量的单克隆抗氨苄青霉素抗体（3H295）和含氨苄青霉素的牛奶样品混合,样品中氨苄青霉素即与抗体结合,然后将该混合样品通入传感器表面,传感器的表面共价固定了氨苄青霉素分子,通过生物特异相互作用分析,检测样品中剩余的抗体,从而得到氨苄青霉素的浓度.样品的测量时间为10min,最低检测限为2.5 ng/mL,低于欧盟标准4 ng/mL.该检测方法的测量时间短、重复性好,批间测量的变异系数为5.4%,表明该方法能满足实际测量要求.     

Label-free detection of leptin antibody-antigen interaction by using LSPR-based optical biosensor

In the recent research, the development of optical biosensing devices has been focused on finding new method and technologies to exploit the optical properties of noble metal nanostructure, especially localized surface plasmon resonance (LSPR). In this study, we fabricated a LSPR-based label-free optical biosensor with the multi-spot gold-capped nanoparticle array (MG-NPA) biochip based on the deposition of a thin gold (Au) film on the silica nanoparticles layer with the simple process. The MG-NPA biochip used the silica nanoparticles as the core and a thin Au film as a shell on the surface. This structure can excite the LSPR signal easily with the high reproducibility. The anti-leptin antibody was immobilized on the surface of MG-NPA biochip, which could recognize only leptin antigen. The leptin antibody-antigen interaction was performed by the introduction of different concentration (1 pg/mL-100 microg/mL) of leptin antigen solutions for 1 h. The detection limit was found to be 100 pg/mL by using the anti-leptin antibody immobilized MG-NPA biochip. This LSPR-based label-free optical biosensor employing the MG-NPA biochip brings several advantages such as low cost, easy to fabricate, using a simple optical system and can be applied in a wide immunoassay with the similar antibody-antigen model.     

Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding microtubules

An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwich immunoassay format. Gliding microtubules carry the antigen-specific "capture" antibody, and bound analyte is detected using a fluorescent viral scaffold as the tracer. A detailed modification scheme for the MTs postpolymerization is described along with corresponding quantification by fluorescence spectroscopy. The resultant antibody-MTs maintain their morphology and gliding capabilities. We report a limit of detection down to 0.5 ng/mL during active transport in a 30 min assay time and down to 1 ng/mL on static surfaces. This study demonstrates the kinesin/MT-mediated capture, transport, and detection of the biowarfare agent SEB in a microfluidic format.     

Cancer biomarker detection in serum samples using surface plasmon resonance and quartz crystal microbalance sensors with nanoparticle signal amplification

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. Here we describe the development of a point-of-care immunosensor for the detection of the cancer biomarker (total prostate-specific antigen, tPSA) using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) sensor platforms in human serum samples. K(D) of the antibody used toward PSA was calculated as 9.46 × 10(-10) M, indicating high affinity of the antibody used in developing the assay. By performing a sandwich assay using antibody-modified nanoparticles concentrations of 2.3 ng mL(-1) (Au, 20 nm) and 0.29 ng mL(-1) (8.5 pM) (Au, 40 nm) tPSA in 75% human serum were detected using the developed assay on an SPR sensor chip. The SPR sensor results were found to be comparable to that achieved using a QCM sensor platform, indicating that both systems can be applied for disease biomarkers screening. The clinical applicability of the developed immunoassay can therefore be successfully applied to patient's serum samples. This demonstrates the high potential of the developed sensor devices as platforms for clinical prostate cancer diagnosis and prognosis.     

Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers

We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules.     

Multidimensional biochemical detection of microcystins in liquid chromatography

The coupling of antibody-, receptor-, or enzyme-based inhibition assays postcolumn to chromatographic systems provides biological detectors with extraordinary high sensitivity and specificity. Three monoclonal antibodies (MC10E7, AD4G2, M8H5) directed against microcystins and protein phosphatase 1 (PP1) were used as off-line detectors for the HPLC separation of microcystins and nodularin in comparison to UV detection. For HPLC/ELISA coupling using antibody MC10E7, a detection limit of 0.04 ng microcystin-LR was achieved. The provisional guideline value for microcystin-LR (1 microg/L, WHO) could be monitored without prior sample concentration, in contrast to UV detection. Quantification of microcystin-LR and two cross-reactants was demonstrated. Furthermore, cross-reactivity or enzyme inhibition of new microcystins, only available in small amounts, can be determined by this method. Using a cyanobacterial extract, HPLC/ELISA coupling was compared to HPLC/UV and electrospray ionization mass spectrometry (ESI-TOFMS).     

Quantification of recombinant interleukin-2 in human serum by a specific immunobioassay

A sensitive and specific assay for recombinant interleukin-2 (rIL-2) in human serum is described. The assay is based on a sequential sandwich immunobioassay that uses a microtiter plate coated with anti-rIL-2 monoclonal antibody (specific for recombinant human IL-2) to capture rIL-2 from serum, and an IL-2 dependent T-cell line that proliferates in a dose-dependent fashion. The lower limit of quantitation of the assay is 2 units/mL (1 unit = approximately 50 pg) using 0.1 mL of serum and the calibration curves ranged from 2 to 50 units/mL. Data are reported on the sensitivity, precision, reproducibility, and specificity of the assay; the stability of rIL-2 in serum; and the recovery of rIL-2 from serum. We also report on the use of the procedure to assay clinical samples from patients with AIDS undergoing treatment with rIL-2.     

Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.     

Versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels

A versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels has been developed for sensitive protein detection. This sandwich-type sensor is fabricated on an indium tin oxide chip covered with a well-ordered gold nanoparticle monolayer. Gel imaging systems were successfully introduced to develop a novel high-efficient optical immunoassay, which could perform simultaneous detection for the samples with a series of different concentrations of a target analyte. The linear range of this assay was between 0.1 and 500 ng/mL, and the assay sensitivity could be further increased to 0.005 ng/mL with the linear range from 0.005 to 100 ng/mL by stripping voltammetric analysis. The immunosensor showed good precision, high sensitivity, acceptable stability, and reproducibility and could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method.