Immunosensor Array Research Articles

Quantitative differentiation of multiple virus in blood using nanoporous silicon oxide immunosensor and artificial neural network

In spite of the rapid developments in various nanosensor technologies, it still remains challenging to realize a reliable ultrasensitive electrical biosensing platform which will be able to detect multiple viruses in blood simultaneously with a fairly high reproducibility without using secondary labels. In this paper, we have reported quantitative differentiation of Hep-B and Hep-C viruses in blood using nanoporous silicon oxide immunosensor array and artificial neural network (ANN). The peak frequency output (fp) from the steady state sensitivity characteristics and the first cut off frequency (fc) from the transient characteristics have been considered as inputs to the multilayer ANN. Implementation of several classifier blocks in the ANN architecture and coupling them with both the sensor chips, functionalized with Hep-B and Hep-C antibodies have enabled the quantification of the viruses with an accuracy of around 95% in the range of 0.04fM–1pM and with an accuracy of around 90% beyond 1pM and within 25nM in blood serum. This is the most sensitive report on multiple virus quantification using label free method.

Chemiluminescence immunoassay for cardiac troponin T by using silver nanoparticles functionalized with hemin/G-quadruplex DNAzyme on a glass chip array

The authors describe an array for chemiluminescence (CL) based determination of cardiac troponin T (cTnT), an important cardiovascular disease marker. The tracing tag consists of silver nanoparticles (AgNPs) loaded with guanine-rich DNA sequences and detection antibody in a high numerical ratio. The loaded AgNPs were then reacted with hemin to form a hemin/G-quadruplex DNAzyme. A disposable immunosensor array was fabricated by immobilizing capture antibody on corresponding sensing sites on a glass chip. Once a sandwich immunocomplex is formed on the array, the tracing tag catalyzes the CL reaction of the luminol-p-iodophenol and H2O2 system to produce a CL signal, which is collected by a CCD camera. An intuitive CL image is obtained containing all of the spots on the array. Under optimal conditions, the method shows a wide linear range over 4 orders of magnitude (from 0.003 to 270 ng·L−1), a detection limit down to 84 fg·L−1, and a throughput as high as 44 tests·h−1. The results obtained with serum samples are in acceptable agreement with reference values. The AgNP-based tracing tag as well as the immunoassay method shows a promising potential for point-of-care testing for the early clinical diagnosis of cardiovascular disease.

An electrode-separated piezoelectric immunosensor array with signal enhancement based on enzyme catalytic deposition of palladium nanoparticles and electroless deposition nickel-phosphorus

A differential piezoelectric immunosensor array was reported with signal enhancement based on the biometallization of palladium nanoparticles (Pd NPs) and successive electroless deposition Ni-P layer. In this strategy, the primary antibody probes were immobilized on the aminated quartz surface in an electrode-separated piezoelectric sensor (ESPS). After bounding with the corresponding antigen and alkaline phosphatase (ALP) conjugated second antibody, the ALP in the sandwich-type immunocomplex can catalyze the substrate of p-aminophenyl phosphate to generate p-aminophenol, which reduces Pd(II) in solution to Pd NPs onto the quartz surface. A successive signal amplification was performed by Pd NPs- promoted catalytic electroless deposition of Ni-P layer. A differential measurement mode was employed to eliminate the influence of non-mass effects, including the changes in conductivity, viscosity, density and temperature of the solution, as well as the baselines draft of piezoelectric sensors. The responses of the differential ESPS in fundamental frequency, third and fifth overtones were compared. The proposed method was applied to the determination of human IgG with a detection limit of 1pg/mL. Compared with biometallization of Pd NPs, the sensitivity is enhanced further by three orders of magnitude due to high activation energy in Ni-P deposition reaction and low baseline draft in differential measurement mode.

Multiplex Immunosensor Arrays for Electrochemical Detection of Cancer Biomarker Proteins.

Measuring panels of protein biomarkers offer a new personalized approach to early cancer detection, disease monitoring and patients' response to therapy. Multiplex electrochemical methods are uniquely positioned to provide faster, more sensitive, point of care (POC) devices to detect protein biomarkers for clinical diagnosis. Nanomaterials-based electrochemical methods offer sensitivity needed for early cancer detection. This review discusses recent advances in multiplex electrochemical immunosensors for cancer diagnostics and disease monitoring. Different electrochemical strategies including enzyme-based immunoarrays, nanoparticle-based immunoarrays and electrochemiluminescence methods are discussed. Many of these methods have been integrated into microfluidic systems, but measurement of more than 2-4 protein markers in a small single serum sample is still a challenge. For POC applications, a simple, low cost method is required. Major challenges in multiplexed microfluidic immunoassays are reagent additions and washing steps that require creative engineering solutions. 3-D printed microfluidics and paper-based microfluidic devices are also explored.

A portable paper-based microfluidic platform for multiplexed electrochemical detection of human immunodeficiency virus and hepatitis C virus antibodies in serum.

This paper presents a portable paper-based microfluidic platform for multiplexed electrochemical detection of antibody markers of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in serum samples. To our best knowledge, this is the first paper-based electrochemical immunosensing platform, with multiplexing and telemedicine capabilities, for diagnosing HIV/HCV co-infection. The platform consists of an electrochemical microfluidic paper-based immunosensor array (E-μPIA) and a handheld multi-channel potentiostat, and is capable of performing enzyme-linked immunosorbent assays simultaneously on eight samples within 20 min (using a prepared E-μPIA). The multiplexing feature of the platform allows it to produce multiple measurement data for HIV and HCV markers from a single run, and its wireless communication module can transmit the results to a remote site for telemedicine. The unique integration of paper-based microfluidics and mobile instrumentation renders our platform portable, low-cost, user-friendly, and high-throughput.

Nanocrystalline Silicon Oxide Impedance Immunosensors for Sub-Femtomolar Mycotoxin Estimation in Corn Samples by Incremental Fuzzy Approach

Detection of trace quantities of common mycotoxins (which have carcinogenic effects) in food samples with sufficient accuracy when present with large quantities of non-target toxins is an existing challenge. To address the problem, in this paper, we have deployed nanocrystalline silicon oxide impedance immunosensor array coupled with incremental fuzzy logic. These sensors have been reported earlier to be more sensitive and specific, compared with others. But toxin estimation becomes sufficiently imprecise especially when the concentration of the non-target toxin is significantly more than that of the target toxin. It is, therefore, expected that this problem will aggravate for real food samples. Thus, to make these sensors commercially viable, the sensor data have been processed by the incremental fuzzy approach. The flexibility in this approach has enabled estimation of three mycotoxins in corn samples procured from two different geographical locations of India. The detection limit achieved is as low as 0.1 fg/ml with 93% accuracy even in presence of large quantities of non-target toxins and with limited calibration data set. In comparison with the existing reports, this is the most sensitive and selective approach for mycotoxin estimation in real food samples with negligible effect of food matrix interference.

Probing molecular interactions with methylene blue derivatized self-assembled monolayers

The emergence of stratified and personalised medicine and the associated need for highly multiplexed detection strategies are driving the development of innovative sensor technology. Electronic immunosensor arrays capable of label-free and highly parallel monitoring of ligand binding have emerged as a particularly promising technology capable of meeting these new diagnostic challenges. In this study, we present an approach for interrogating molecular interactions electronically using redox active molecular monolayers. Specifically, we have synthesised self-assembled molecular monolayers assembled from long-chain alkanethiols (LCAT) incorporating oligoethyleneglycol (OEG) linkers that can be derivatized with a range of functional groups, including the redox active molecule methylene blue. Critically, we show that the electron transport properties of this redox-active monolayer are highly sensitive to the electrochemical environment, including the local concentration of protons and the electrostatic potential at the plane of electron transfer. Using a combination of cyclic voltammetry and QCM-D to study in detail the behaviour of the monolayer during functionalisation and analyte binding, we demonstrate that these redox properties can be exploited for the electrochemical sensing of molecular interactions (biotin–avidin in our case) on SAMs. Given the versatility of LCAT-OEG monolayers, in terms of linker lengths, choice of functional group, and ability to create mixed component layers and the straight-forward assembly of mixed SAMs of high quality, our electrochemical sensing approach forms an excellent and generic label-free platform for probing a wide range of molecular interactions.

Multiplexed electrochemical immunoassay using streptavidin/nanogold/carbon nanohorn as a signal tag to induce silver deposition

An ultrasensitive multiplexed immunoassay method was developed by using streptavidin/nanogold/carbon nanohorn (SA/Au/CNH) as a novel signal tag to induce silver enhancement for signal amplification. The Au/CNH was prepared by in situ growth of nanogold on carboxylated CNH and functionalized with streptavidin. The SA/Au/CNH showed well dispersibility in physiological buffer and could sever as a common tracing tag to recognize biotinylated signal antibody. The immunosensor array was prepared on disposable screen-printed electrodes. Through sandwich-type immunoreaction and biotin-streptavidin affinity reaction, the SA/Au/CNH tag was captured on the immunoconjugates to induce silver deposition and amplify the electrochemical stripping signals. Using α-fetoprotein and carcinoembryonic antigen as model analytes, the proposed method showed wide linear ranges with the detection limits down to 0.024pgmL−1 and 0.032pgmL−1, respectively, and eliminated completely signal cross-talk between adjacent immunosensors. It provided a convenient, high-efficient and ultrasensitive electrochemical detection route for biological analytes, showing great potential in clinical application.

A novel dual-template molecularly imprinted electrochemiluminescence immunosensor array using Ru(bpy)32+-Silica@Poly-L-lysine-Au composite nanoparticles as labels for near-simultaneous detection of tumor markers

A novel electrochemiluminescence (ECL) immunosensor array was fabricated on a screen-printed electrode (SPE) to perform multiplexed immunoassay of tumor markers (TMs), such as carcinoembryonic antigen (CEA) and carbohydrate antigen-199 (CA199). The SPE substrate consisted of a common Ag/AgCl reference electrode, a common carbon counter electrode and two gold nanoparticles modified carbon working electrodes. Firstly, dopamine (DA) as the functional monomer along with the corresponding TMs (CEA or CA199) as template molecules was electro-polymerized on the surface of different working electrodes. Then, the dual-template molecularly imprinted polydopamine (MIP-PDA) film was formed as the capture probe to recognize CEA and CA199. Secondly, a novel label was fabricated by conjugating the antibodies of TMs with Ru(bpy)32+-Silica@Poly-L-lysine-Au (Ru-Si@PLL-Au) nanocomposites, in which gold colloids (Au NPs) were doped on the surface of Ru-Silica (Ru(bpy)32+-doped Silica) using poly-L-lysine (PLL) as a bridging agent. PLL was also employed as a co-reactant of the luminophore. Based on a sandwich-type immunoassay, the MIP-PDA film, the corresponding antigen and labels were conjugated to produce the immunocomplex. The signals from the ECL immunosensor array were near-simultaneously detected by a photomultiplier tube (PMT) using a homemade single-pore-two-throw switch, which could avoid crosstalk between adjacent working electrodes. The obtained concentrations were 0.05-100pgmL−1 for CEA and 0.03-80 U L−1 for CA199, with detection limits of 0.02pgmL−1 and 0.01 U L−1, respectively. This novel ECL strategy provides a simple, economical, fast and sensitive approach for multiplexed immunoassay of CEA and CA199, and has significant potential for protein detection in a clinical laboratory setting.

A nanowire-based label-free immunosensor: Direct incorporation of a PSA antibody in electropolymerized polypyrrole

We have suggested a novel method for the preparation of a label-free electrochemical immunosensor for the detection of prostate-specific antigen (PSA) as target marker for prostate cancer. Direct incorporation of PSA antibody (anti-PSA) into polypyrrole (Ppy) electropolymerized on a three-dimensional Au nanowire array has resulted in enhanced molecular interactions, ultimately leading to improved sensing performance. The electrochemical performance of the nanowire-based immunosensor array were characterized by (1) differential pulse voltammetry (DPV) to evaluate the specific recognition of PSA, (2) impedance and cyclic voltammetry to observe surface resistance and electroactivity, and (3) scanning electron microscopy (SEM) to demonstrate the three-dimensional architecture. The vertically-aligned geometric organization of Ppy provides a novel platform to improve the anti-PSA loading capacity. Overall, enhanced electrochemical performance of the proposed immunosensor has been demonstrated by its linear response over PSA concentrations ranging from 10fgmL−1 to 10ngmL−1 and a detection limit of 0.3fgmL−1, indicating that the strategy proposed here has great potential for clinical applications.

Ultrasensitive electrochemical immunosensors for multiplexed determination using mesoporous platinum nanoparticles as nonenzymatic labels

An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab2) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab1) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab2 was bound to immunosensor surface to catalyze the electro-reduction of H2O2 reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002UmL−1, 0.001UmL−1 and 7.0pgmL−1 for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics.

Development and validation of a novel leaky surface acoustic wave immunosensor array for label-free and high-sensitive detection of cyclosporin A in whole-blood samples

This manuscript described a novel 2×3 model of leaky surface acoustic wave (LSAW) immunosensor array for label-free and high-sensitive detection of Cyclosporin A (CsA) in whole-blood samples. In this technique, every resonator crystal unit of the LSAW immunosensor array had an individual oscillator circuit to work without mutual interference. The LSAW immunosensor was first immobilized with protein A from Staphylococcus aureus and monoclonal anti-CsA antibody on the gold electrode surface of 100MHz LiTaO3 piezoelectric crystals, which then captured the CsA. The CsA increased the mass loading of LSAW immunosensor and leaded to phase shifts of LSAW. Consequently, under optimal conditions, the designed LSAW immunosensor exhibited a detection limit of 0.89ng/mL, quantification limit of 2.96ng/mL, and wide dynamic linear range from 1ng/mL to 1000ng/mL for CsA detection. Application of the LSAW immunosensor array to clinical sample revealed that consistency and comparability between LSAW immunosensor and the enzyme multiplied immunoassay method were good. Moreover, the immunosensor could be regenerated for ten times without appreciable loss of activity. Therefore, the self-designed LSAW immunosensor array provided a rapid, accurate, label-free, easy handling, and dynamic real-time method for the detection of immunosuppressive drugs in clinical laboratory.

A multianalyte electrochemical immunosensor based on patterned carbon nanotubes modified substrates for detection of pesticides

A novel multianalyte electrochemical immunosensor based on the assembly of patterned SWNTs on glassy carbon (GC) substrates was developed for simultaneous detection of endosulfan and paraoxon. Based on aryldiazonium salt chemistry, forest of SWNTs can be patterned on GC substrates by C3C bonding using micro contact printing (MCP), which provides an interface showing efficient electron transfer between biomolecules and electrodes. Then redox molecules FDMA and PQQ can be attached to the SWNTs, respectively followed by the attachment of specific epitopes and antibodies. The modified sensing surfaces were characterized by XPS, SEM, AFM and electrochemistry. Based on the current change of specific redox probes, the fabricated immunosensor array can be used for simultaneous detection of endosulfan and paraoxon by a displacement assay. In phosphate buffer solution (50mM, pH 7.0), there is a linear relationship between electrochemical signal of FDMA and the concentration of endosulfan over the range of 0.05–100ppb with a detection limit of 0.05ppb; the linear range between electrochemical signal of PQQ and the concentration of paraoxon is 2–2500ppb with a detection limit of 2ppb. The immunosensor array demonstrates high repeatability, reproducibility, stability and selectivity for the detection of endosulfan and paraoxon.

Electrochemical immunoassay for procalcitonin antigen detection based on signal amplification strategy of multiple nanocomposites

Procalcitonin, as a medium of inflammation, has become a new marker of the identification of severe bacterial infections in recent years and has received high attention due to its most ideal diagnostic indicators of specificity with major types of organism systemic inflammation of bacterial infection in the early stages. Thus, a novel method for the determination of procalcitonin (PCT) was developed based on a sandwich-type electrochemical immunosensor, which combined a simple immunosensor array as well as an effectively designed trace tag. The immunosensor was fabricated by layer-by-layer coating graphene (GC), carbon nanotubes (MWCNTs), chitosan (CS), glutaraldehyde (GA) composite on the working electrode, which can increase the electronic transfer rate and improve the surface area to capture a large number of primary antibodies (Ab1). The trace tag was prepared by loading high-content signal horseradish peroxidase labeled secondary PCT antibody (HRP-Ab2) with AuNPs, which were coated with mesoporous silica nanoparticles (MCM-41) through thionine linking. In comparison with conventional methods, the proposed immunosensor for PCT provided a better linear response range from 0.01 to 350ng/mL and a lower limit of detection (LOD) of 0.5pg/mL under optimal experimental conditions. In addition, the immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of PCT in real human serum samples, indicating that the developed immunoassay has the potential to find application in clinical detection of PCT and other tumor markers as an alternative approach.

Double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor

A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at −0.25V in 0.10M PBS (pH 7.0) containing 0.1mM hydroquinone and 2.0mM H2O2. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0×10−10gmL−1 to 1.0×10−8gmL−1 with a detection limit of 3×10−11gmL−1. Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.

Silver nanowire-based electrochemical immunoassay for sensing immunoglobulin G with signal amplification using strawberry-like ZnO nanostructures as labels

The quick development of nanoscience and nanotechnology has paved the way for ultrasensitive biosensing and analysis. In this work, an ultrasensitive electrochemical immunosensor was developed for the detection of human immunoglobulin G (IgG) by combining with a newly designed trace tag on a disposable immunosensor array. The array was prepared by immobilizing captured antibodies on ultralong Ag nanowires, whilst the trace tag was prepared by loading horseradish peroxidase (HRP)-labeled goat anti-human IgG (HRP-anti-IgG) on thionine (TH)-doped mesoporous ZnO nanostrawberries (MP-ZnO). With a sandwich-type immunoassay format, mainly due to crystalline framework and high surface area of the mesoporous (MP) materials, as well as the superconductivity of silver nanowires, the electrochemical signal was significantly amplified. The linear range of the developed immunosensor is 0.01–200ngmL−1 and the detection limit is 4pgmL−1 IgG, which make the hierarchically nanostructured composites very promising candidates for the next-generation sandwich-type electrochemical immunoassays.

Diagnosis of schistosomiasis japonica with interfacial co-assembly-based multi-channel electrochemical immunosensor arrays

Schistosomiasis control remains to be an important and challenging task in the world. However, lack of quick, simple, sensitive and specific sero-diagnostic test is still a hurdle in the control practice. The commonly employed enzyme-linked immuno-sorbent assay (ELISA) relies on the native soluble egg antigen (SEA) that is limited in supply. Here we developed an electrochemical immunosensor array (ECISA) assay with an interfacial co-assembly strategy. A recombinant Schistosoma japonicum (Sj) calcium-binding protein (SjE16) was used as a principal antigen, while the SEA as a minor, co-assembling agent, with a ratio of 8:1 (SjE16: SEA, Sj16EA), which was co-immobilized on a disposable 16-channel screen-printed carbon electrode array. A portable electrochemical detector was employed to detect antibodies in serum samples. The sensitivity of ECISA reached 100% with minimal cross-reactions. Therefore, we have demonstrated that this rapid, sensitive and specific ECISA technique has the potential to perform large-scale on-site screening of Sj infection.

A disposable electrochemical immunosensor arrays using 4-channel screen-printed carbon electrode for simultaneous detection of Escherichia coli O157:H7 and Enterobacter sakazakii

An electrochemical immunosensor for Escherichia coli O157:H7 (E. coli O157:H7) and Enterobacter sakazakii (E. sakazakii) detection using carbon screen-printed low-density arrays is reported. The sensors were fabricated based on screen-printed carbon arrays containing four carbon working electrode, an integrated carbon counter electrodes and an integrated Ag/AgCl reference electrode. Multi-walled carbon nanotubes (MWCNTs)/sodium alginate (SA)/carboxymethyl chitosan (CMC) composite films were coated on all the working electrodes to enhance the sensitization of the electrode. Horseradish peroxidases (HRP) labeled antibodies of two bacteria were immobilize on different working electrode of the same screen-printed electrode respectively. The immobilization of MWCNTs, HRP labeled antibodies onto the screen-printed carbon electrodes was examined using atom force microscopy (AFM) and cyclic voltammetry (CV). The analytical performance of proposed immunosensor arrays toward E. sakazakii and E. coli O157:H7 was investigated by AFM and CV. Under optimal conditions, the linear range of E. sakazakii and E. coli O157:H7 were from 104 to 1010cfu/ml, with a detection limit of 4.57×103cfu/ml (S/N=3) and 3.27×103cfu/ml (S/N=3), respectively. The specificity, reproducibility, stability and accuracy of the proposed immunosensor arrays were also evaluated. Two antibodies modified work electrodes were tested and compared in terms of sensitivity and ability to recognize different pathogenic biological species.

Ultrasensitive multiplexed immunosensors for the simultaneous determination of endocrine disrupting compounds using Pt@SBA-15 as a non-enzymatic label.

The simultaneous determination of different analytes based on multiplexed immunosensors is attractive and promising in bioassays and environmental analysis. In this paper, a novel ultrasensitive multiplexed immunoassay method was developed for the determination of endocrine disrupting compounds (EDCs) based on disposable screen-printed carbon electrodes. Different capture antibodies were separately immobilized on the carbon electrodes to form an immunosensor array. Platinum nanoparticle-functionalized mesoporous silica nanoparticles (Pt@SBA-15) was used as catalytic labels of secondary antibodies. Due to the catalytic activities of the platinum nanoparticles and the large surface area of SBA-15, a strong electrical response towards the analytical antigens was achieved. The multiplexed immunoassay array enables the simultaneous determination of different analytes. Using diethylstilbestrol and estradiol as model analytes, this multiplexed immunoassay method showed wide linear ranges with detection limits down to 0.28 and 1.2 pg mL-1 for diethylstilbestrol and estradiol, respectively. The proposed sensing strategy enriches electrochemical immunoassays and has a promising application in bioassays and environmental analysis.

A disposable immunosensor device for point-of-care test of tumor marker based on copper-mediated amplification

A paper-based immunodevice was fabricated for point-of-care test (POCT). The array device was simple and easily assembled. It comprised as many as 4×10 detection points on a single paper array. The immunosensor array was prepared by covalently immobilizing capture antibodies on corresponding working zone on a disposable paper array. With a sandwich-type immunoreaction, the CuO nanoparticles (CuO NPs)-labeled secondary antibody (Ab2) bioconjugates were captured in each working zone. The coordination of dithizone (DZ) at the surface of CdTe quantum dots (CdTe QDs) could strongly quench the green emission of CdTe QDs by a fluorescence resonance energy transfer (FRET) mechanism. After the Cu2+ was released from CuO NPs-Ab2, the fluorescence of CdTe QDs–DZ was “turn-on”. The fluorescence intensity would increase with the increasing of analytes. The calibration plot showed a good linear relationship between the fluorescence intensity and the logarithm value of the analytes concentration with the low detection limit. The immunosensor array was performed for cancer screening. The high throughput, low-cost, acceptable stability, reproducibility, sensitivity and accuracy showed good applicability of the proposed multiplex immunoassay in clinical diagnosis. The results indicated that the device could be applied to comprehensive sample and point-of-care detection.