Ultrasensitive Detection Of Cancer Biomarkers Research Articles

ReS2-MoS2 heterojunction nanotube FET with superlattice structures for ultrasensitive detection of miRNA

Ultrasensitive detection of cancer biomarkers holds immense importance in facilitating the early diagnosis and treatment of cancer. Compared to conventional detection techniques, field-effect transistor (FET) biosensors have demonstrated substantial potential in achieving sensitive detection of cancer biomarkers. However, the ultrasensitive FET still remains great challenges due to the limited electron transport capacity and sensitivity of channel materials. Herein, we employed rhenium disulfide-molybdenum disulfide nanotube (ReS2-MoS2 NT) featuring superlattice structures as channel materials in FET biosensors for the highly sensitive detection of cancer biomarker miRNA-21. More concretely, ReS2-MoS2 NT featuring superlattice structures was synthesized using the anodic aluminum oxide (AAO) template sacrifice method and atomic layer deposition (ALD) method. The electrical properties of single ReS2-MoS2 NT FET were regulated by adjusting the total layers and number of heterojunction interfaces, and the optimal number of heterojunction interfaces of 7 was obtained. Finally, FET biosensors utilizing the network ReS2-MoS2 NT as the channel materials were fabricated and exhibited remarkable sensitivity in detecting miRNA-21 under an external light source. The linear range for detection spanned from 10 aM to 1 nM, with an exceptionally low detection limit of 2.1 aM. This study holds promise for replacing current channel materials and surpassing the detection threshold of cancer biomarkers in the future.

An Electrochemical Immunoassay System for Measuring Circulating Protein Biomarkers of Pediatric Soft Tissue Sarcoma

The combination of simple, inexpensive 3D-printed flow cells with electrochemical biosensors has recently emerged a promising strategy for detecting molecular biomarkers of disease. In this study, we report a multiplex electrochemical immunoassay based on pencil graphite electrode arrays, 3D-printed flow cells and magnetic bead bioconjugates for simultaneous detection of three biomarker proteins (cancer antigen 125 (CA-125), midkine (MK) and osteopontin (OPN)) linked to pediatric soft tissue sarcoma (PSTS). Magnetic bead bioconjugates are functionalized with large amounts of antibody and enzyme labels according to a previously reported strategy,1 pencil graphite electrodes are modified with gold nanoparticles and antibodies for specific capture of bioconjugate-labeled biomarkers, and 3D-printed flow cells to facilitate amperometric detection. Using this immunoassay platform, detection limits for CA-125, MK and OPN that are 100 times lower than those obtained using commercial enzyme-linked immunosorbent assay (ELISA) can be achieved. The combination of low-cost pencil graphite electrode arrays with the convenience of multi-labeled magnetic bead bioconjugates and simplicity of 3D-printed flow-cells results in a promising electrochemical biosensing strategy for biomarker proteins CA-125, MK, and OPN in human serum samples.Reference(1) Malhotra, R.; Patel, V.; Chikkaveeraiah, B. v.; Munge, B. S.; Cheong, S. C.; Zain, R. B.; Abraham, M. T.; Dey, D. K.; Gutkind, J. S.; Rusling, J. F. Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array. Analytical Chemistry 2012, 84 (14), 6249–6255. https://doi.org/10.1021/ac301392g.

Elucidating Raman Image-Guided Differential Recognition of Clinically Confirmed Grades of Cervical Exfoliated Cells by Dual Biomarker-Appended SERS-Tag.

Ultrasensitive detection of cancer biomarkers via single-cell analysis through Raman imaging is an impending approach that modulates the possibility of early diagnosis. Cervical cancer is one such type that can be monitored for a sufficiently long period toward invasive cancer phenotype. Herein, we report a surface-enhanced Raman scattering (SERS) nanotag (SERS-tag) for the simultaneous detection of p16/K-i67, a dual biomarker persisting in the progression of squamous cell carcinoma of human cervix. A nanoflower-shaped SERS-tag, constituted of hybrid gold nanostar with silver tips to achieve maximum fingerprint enhancement from the incorporated reporter molecule, was further functionalized with the cocktail monoclonal antibodies against p16/K-i67. The recognition by the SERS-tag was first validated in cervical squamous cell carcinoma cell line SiHa as a foot-step study and subsequently implemented to different grades of clinically confirmed exfoliated cells including normal cell (NC), high-grade intra-epithelial lesion (HC), and squamous cell carcinoma (CC) samples of the cervix. Precise Raman mapped images were constituted based on the average intensity gradient of the signature Raman peaks arising from different grades of exfoliated cells. We observed a distinct intensity hike of around 10-fold in the single dysplastic HC and CC samples in comparison to NC specimen, which clearly justify the prevalence of p16/Ki-67. The synthesized probe is able to map the abnormal cells within 20 min with high reproducibility and stability for 1 mm × 1 mm mapping area with good contrast. Amidst the challenges in Raman image-guided modality, the technique was further complemented with the gold standard immunocytochemistry (ICC) dual staining analysis. Even though both are time-consuming techniques, tedious steps can be avoided and real-time readout can be achieved using the SERS mapping unlike immunocytochemistry technique. Therefore, the newly developed Raman image-guided SERS imaging emphasizes the approach of uplifting of SERS in practical utility with further improvement for clinical applications for cervical cancer detection in future.

Layer-by-layer self-assembly of MoS2/PDDA hybrid film in microfluidic chips for ultrasensitive electrochemical immunosensing of alpha-fetoprotein

Ultrasensitive detection of cancer biomarkers holds great promise for early stage detection and diagnosis of cancer. Microfluidic electrochemical immunochips have been assessed as powerful detection platforms due to their high sensitivity, low cost, portability, and ease of miniaturization. In this work, an ultrasensitive electrochemical immunochip based on MoS2/PDDA hybrid film is designed via layer-by-layer self-assembly method. An electrochemical impedance detection platform is developed for the detection of alpha-fetoprotein (AFP) by integrating a three-electrode system in the micro-channel, which introduces MoS2/PDDA film modified with antibodies as the working electrode, Ag/AgCl wire as the reference electrode and indium tin oxide (ITO) as the counter electrode. After MoS2/PDDA film is modified with AFP antibodies, the immobilization of various concentrations of AFP antigens is carried on which can cause changes in the electrochemical impedance value. This proposed approach exhibits a linear range from 0.1 ng/mL to 10 ng/mL and a low detection limit of 0.033 ng/mL (S/N = 3). The applicability of the MoS2/PDDA immunochip is further investigated by detecting human serum samples. This ultrasensitive microfluidic immunochip is proved to be highly selective with small input liquid volume, which could realize trace detection of AFP and be potentially applied in clinical diagnosis of other biomolecules.

Abstract B60: Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma

Abstract The last few years have witnessed exceptional advances in liquid biopsies based on the isolation and analysis of diverse tumor-derived material circulating in the blood, such as cell-free DNA (cfDNA) and extracellular vesicles (EVs). However, the isolation of different tumor-derived circulating components from a single aliquot of patients’ plasma and the consequent accurate detection of cancer biomarkers is still challenging. By taking advantage of a recently published nickel-based EVs isolation (NBI) protocol (1) that allows for recovery of cfDNA after EVs isolation, we generated a high-sensitivity molecular assay to accurately detect ERBB2 amplification and consequent HER2 overexpression on a limited volume of plasma collected from 20 breast cancer patients (stage I, II, and III) at diagnosis. The amplification of ERBB2 gene and the overexpression of the relevant encoded protein HER2 are currently used in the clinic for breast cancer classification at diagnosis and subsequent treatment decision with the anti-HER2 monoclonal antibody trastuzumab. By performing droplet digital PCR (ddPCR), we were able to detect ERBB2 amplifications in plasma cfDNA from 20 patients selected based on in situ evaluation of tissue biopsies (IHC and FISH). Moreover, we set up an antibody-based affinity reaction to detect HER2 protein on the surface of the isolated EVs that confirmed HER2 overexpression on a subset of patients. Most importantly, we also succeeded in absolute quantification of HER2 transcripts enclosed within EVs by performing ddPCR in samples of patients showing a range of circulating tumor-derived material. The yields and detection performance of this novel methodologic assay were tested on a cohort of healthy individuals (n=20) and on a cohort of triple-negative (and therefore HER2-negative) breast cancer patients (TNBC; n=20; stage: I, II, III). Overall, here we report a pilot study on a novel multimodal noninvasive method for breast cancer-specific biomarker detection from a minimal amount of plasma (1,5mL) integrating cfDNA-derived information with EVs-derived RNA and proteins analysis. This proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. Reference: 1. Notarangelo M et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine 2019;43:114–26. Citation Format: Vera Mugoni, Caterina Nardella, Orsetta Quaini, Yari Ciani, Michela Notarangelo, Antonella Ferro, Orazio Caffo, Vito D'Agostino, Francesca Demichelis. Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B60.

Rapid, quantitative and ultra-sensitive detection of cancer biomarker by a SERRS-based lateral flow immunoassay using bovine serum albumin coated Au nanorods.

Early diagnosis of cancer biomarkers is the key to guiding treatments and improving the survival rate of patients. Herein, we report a novel surface-enhanced resonance Raman scattering (SERRS)-based lateral flow immunoassay (LFIA) for quantitative and ultra-sensitive analysis of alpha-fetoprotein (AFP). Gold nanorods (AuNRs) were fabricated to be in resonance with 785 nm laser excitation, that is, the excitation level that can maximize SERRS activity. The AuNRs were modified with 5,5′-dithiobis(2-nitrobenzoic acid), bovine serum albumin (BSA), and AFP detection antibody successively as the SERRS nanotags for the LFIA system. Modification of the BSA layer guaranteed good stability and biocompatibility of the SERRS nanotags in complex samples. The SERRS-LFIA strip for AFP detection showed a low detection limit of 9.2 pg mL−1 and a broad detection range from 10 pg mL−1 to 500 ng mL−1. By comparison, the detection limit of the proposed assay is about 100 and 10 times lower than those of the Au nanoparticle-based SERS-strip and conventional enzyme-linked immunosorbent assay, respectively. Moreover, the potential clinical applications of the assay were evaluated by detecting 10 actual serum samples. Results showed 100% accuracy based on the clinical tests.

Biosynthesized Quantum Dot for Facile and Ultrasensitive Electrochemical and Electrochemiluminescence Immunoassay.

Nanomaterials are commonly utilized for amplified immunoassay of biomarkers. However, traditional nanomaterial-based immunoassay usually requires a time-consuming and labor-intensive nanoparticle modification and conjugation process, which impedes their practical applications. Here, a new immunoassay method based on biosynthesized nanomaterials is developed with versatile functions for facile and ultrasensitive detection of cancer biomarker. In this method, the utilized biosynthesized quantum dots (BQDs) allow convenient antibody conjugation and electrode modification, and demonstrate excellent electrochemical and electrochemiluminescent responses. The differential pulse voltammetric, faradaic impedance spectroscopy, and electrochemiluminescent measurements with the BQD-modified electrode show detection limits at picomolar levels as well as good specificity toward human prostate-specific antigen detection. The inherent recognization capability as well as the inherent electrochemical and electrochemiluminescence features thus enable BQDs as good candidates for facile immunosensors with high sensitivity. Such a biosynthesized nanomaterial-based approach opens up the possibility of using innovative designs for nanoparticle-based assays, and developing reliable and practical methods for early disease diagnosis.

3D DNA nanosphere-based photoelectrochemical biosensor combined with multiple enzyme-free amplification for ultrasensitive detection of cancer biomarkers

In this work, a new 3D DNA nanosphere was ingeniously designed and fabricated, which was used to combine with multiple enzyme-free amplification strategy to develop a photoelectrochemical (PEC) biosensing platform for ultrasensitive detection of carcinoembryonic antigen (CEA). The 3D DNA nanostructure was self-assembled by base complementary pairing in a few minutes and rolling circle amplification (RCA) reaction. The intense photocurrent derived from Au NPs/ZnSe QDs can be effectively decreased by 3D DNA nanospheres assembled on the electrode, making photoelectric signal present “off” state. The specific binding of target CEA with its hairpin (HP1) aptamer opens HP1 structure, which initiated multiple enzyme-free strand displacement amplification (SDA) reaction and generated a large number of single strands DNA S1. Then S1 competitively binds to capture DNA on the electrode to release 3D DNA nanospheres, thus the photocurrent signal became “on” state for achieving amplified assay of target CEA. The proposed PEC biosensor exhibits excellent performance with a wide linear range of 1.0 fg/mL to 10 ng/mL and a low detection limit of 0.12 fg/mL for CEA, which was successfully applied for the assay of real serum samples with good precision. The reported strategy opens a new simple way for PEC biosensor using DNA nanostructure, showing huge potential in clinical application research.

Silicon Nanowires Field Effect Transistors: A Comparative Sensing Performance between Electrical Impedance and Potentiometric Measurement Paradigms.

Potentiometric sensors based on silicon nanowire field effect transistors (SiNW FETs) typically display exquisite sensitivities, but their bioanalytical implementation is limited due to the need for stringent measurement conditions and high-precision readout units. An alternative operation principle where SiNW FETs are operated in a frequency-domain electrical impedimetric approach is promising. However, to date only limited data is available in regard to the sensing performance and translational relevance of this novel approach in comparison to the standard charge detection paradigm. We demonstrate the feasibility of conducting electrical impedimetric FET measurements with a portable unit for the ultrasensitive detection of cancer biomarkers in biospecimens. Compared to standard potentiometric measurements, electrical impedimetric FET measurements yielded significant improvements in biosensing performances, including the limit of detection, sensing resolution, and dynamic range.

Ultrasensitive analysis of carcinoembryonic antigen based on MoS2-based electrochemical immunosensor with triple signal amplification

Ultrasensitive detection of cancer biomarkers has shown great promise for precision medicine. Here, a triple signal amplification strategy was developed for analysis of carcinoembryonic antigen (CEA) by using MoS2-based nanocomposites. Gold nanoparticles-decorated molybdenum disulfide nanocomposite (MoS2-AuNPs) was used to construct the modified electrode and nanoprobe, which could efficiently amplify electrochemical signal due to its large surface area and high catalytic ability. Horseradish peroxidase (HRP)-labelled carcinoembryonic monoclonal antibody (anti-CEA) and HRP were used to co-construct the MoS2-based nanoprobe, which could further amplify the electrochemical signal by catalyzing o-phenylenediamine (o-PD) in the presence of hydrogen peroxide (H2O2). Expectedly, an excellent analytical performance for CEA detection was obtained, such as wide detection range (10 fg mL−1-1 ng mL−1), ultralow detection limit (1.2 fg mL−1), high selectivity and good stability, suggesting this immunosensor could detect CEA in real samples.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BackgroundExtracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice.MethodsWe designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility.FindingsFrom plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies.InterpretationWe propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies.FundAssociazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Biofunctionalized two-dimensional Ti3C2 MXenes for ultrasensitive detection of cancer biomarker

In this work, ultrathin Ti3C2-MXene nanosheets were synthesized by minimally intensive layer delamination methods, and uniformly functionalized with aminosilane (f-Ti3C2-MXene) to provide a covalent binding for the immobilized bio-receptor (anti-CEA) for label free, ultrasensitive detection of cancer biomarker (carcinoembryonic antigen, CEA). The effect of different redox probes on the electrochemical behavior of f-Ti3C2-MXene was investigated and found that hexaammineruthenium ([Ru(NH3)6]3+) is the preferable redox probe for biosensing. The fabricated biofunctionalized Ti3C2-MXene exhibits a linear detection range of 0.0001–2000 ng mL−1 with sensitivity of 37.9 µA ng−1 mL cm−2 per decade. The wider linear detection range of our f-Ti3C2-MXene is not only higher than previously reported pristine 2D nanomaterials, but is even comparable to other hybrid 2D nanomaterials. We believe that this work opens a new window for development of MXene-based highly sensitive DNA, aptamer, enzyme, antibody, and cell based biosensors, and could be further used in drug delivery application.

Ternary Electrochemiluminescence Nanostructure of Au Nanoclusters as a Highly Efficient Signal Label for Ultrasensitive Detection of Cancer Biomarkers.

Herein, the Au nanoclusters (Au NCs) functionalized ternary nanostructure with significant electrochemiluminescence (ECL) emission was first proposed to fabricate an ultrasensitive immunosensor for carcinoembryonic antigen (CEA) detection, which was established by combining bovine serum albumin (BSA) templated Au NCs as the luminophor, tris(3-aminoethyl)amine (TAEA) as the coreactant, and Pd@CuO nanomaterial as the coreaction accelerator via covalent attachment in a nanostructure. Through the dual self-catalysis including intramolecular coreaction between TAEA and Au NCs, and intramolecular coreaction acceleration from Pd@CuO to TAEA, the Au NCs-TAEA-Pd@CuO achieved excellent ECL performance so that the detection limit of the immunosensor for measuring CEA antigen was down to 16 fg/mL in the absence of any additional signal amplification assay. Moreover, the method for preparing ECL nanostructure-based metal NCs should be devoted to the development of highly efficient ECL signal label, outlining a significant scheme toward biological testing and clinical diagnosis.

Ultrasensitive detection of cancer biomarkers using conducting polymer/electrochemically reduced graphene oxide-based biosensor: Application toward BRCA1 sensing

Breast Cancer (BRCA) is the most common threat in women worldwide. Increasing death rate of diagnosed cases is the main leading cause of designing specific genosensors for BRCA − related cancer detection. In the present study, an ultrasensitive label − free electrochemical DNA (E − DNA) sensor based on conducting polymer/reduced graphene − oxide platform has been developed for the detection of BRCA1 gene. An electrochemical method was applied as a simple and controllable technique for the electrochemical reduction of graphene oxide and also, electro − polymerization of pyrrole − 3 − carboxylic acid monomer. The results of the present work show that the polymer − coated reduced graphene − oxide provide more active sites compare to polymer − coated glassy carbon electrode for the DNA probe immobilization. The fabricated signal − off E − DNA sensor employs Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) techniques for monitoring the electrochemical behavior of the redox probe. To survey the morphological pattern and surface structural characterizations, the Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) have been applied, respectively. The applicability of the modified surface layer toward DNA sensing was evaluated by both DPV and EIS measurements. This platform permits quantitative determination of BRCA1 in the range of 10 fM–0.1 μM with a limit of detection as low as 3 fM. The proposed genosensor showed a perfect discriminatory power between complementary, non − complementary and mismatched DNA sequences. The described method combined the outstanding features in terms of selectivity, sensitivity, repeatability, reproducibility and remarkable reusability, without demanding for labor − intensive labeling steps. Moreover, the modified electrode was successfully used for accurate determination of trace amounts of DNA target in blood plasma samples.

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Dual-responsive immunosensor that combines colorimetric recognition and electrochemical response for ultrasensitive detection of cancer biomarkers

We developed a nanoroughened, biotin-doped polypyrrole immunosensor for the detection of tumor markers through dual-signal (electrochemical and colorimetric) channels, electrochemical and colorimetric, that demonstrates remarkable analytical performance. A rapid, one-step electric field-mediated method was employed to fabricate the immunosensor with nanoscale roughness by simply modulating the applied electrical potential. We demonstrated the successful detection of three tumor markers (CA125, CEA, and PSA) via the double enzymatic signal amplifications in the presence of a target antigen, ultimately leading to desired diagnostic accuracy and reliability. The addition of multiple horseradish peroxidase (HRP)- and antibody-labeled nanoparticles greatly amplified the signal and simplified the measurement of cancer biomarker proteins by sequentially magnifying electrochemical and colorimetric signals in a single platform. The two parallel assays performed using the proposed immunosensor have yielded highly consistent and reproducible results. Additionally, for the analysis of plasma samples in a clinical setting, the values obtained with our immunosensor were validated by correlating the results with those of a standard radioimmunoassay (RIA), which obtained very similar clinically valid responses.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ngmL−1, and the half of saturation concentration (SC50) value was 6.68±0.56ngmL−1. These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ngmL−1, and the linear range was 0.01–10,000ngmL−1. These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

A versatile immobilization-free photoelectrochemical biosensor for ultrasensitive detection of cancer biomarker based on enzyme-free cascaded quadratic amplification strategy.

In this work, an ultrasensitive immobilization-free photoelectrochemical (PEC) biosensor was successfully developed for the first time based on a novel enzyme-free cascaded quadratic signal amplification strategy. This rationally designed homogeneous dual amplification strategy consists of a target-analog recycling circuit based on catalytic hairpin assembly (CHA) and a hybridization chain reaction (HCR) mediated amplification circuit. In the presence of carcinoembryonic antigen (CEA), a proof-of-concept target, target-analog is released to trigger the upstream CHA recycling circuit. The generated dsDNA complexes from CHA recycling could further induce the downstream HCR amplification, leading to the formation of numerous hemin/G-quadruplex DNAzymes. This would accordingly stimulate the biocatalytic precipitation of 4-chloro-1-naphthol, inducing a distinct decrease in the photocurrent signal due to the formed insoluble/insulating products on electrode surface. Under the optimal conditions, this PEC biosensor achieved ultrasensitive detection of CEA down to the atto-gram level. The introduction of this aptamer-based cascaded quadratic amplification strategy not only remarkably improves the selectivity and sensitivity of CEA assay, but also allows the ultrasensitive detection of other proteins by designing specific aptamers, providing a universal, isothermal and label-free PEC biosensing platform for ultrasensitive detection of different kinds of cancer biomarkers and holding a great potential for early-diagnosis of cancer.

Au nanoparticles/PAMAM dendrimer functionalized wired ethyleneamine–viologen as highly efficient interface for ultra-sensitive α-fetoprotein electrochemical immunosensor

In this work, a novel electrochemical immunoassay system is developed for ultrasensitive detection of cancer biomarker, α-fetoprotein (AFP). This immunosensor is constructed by covalent immobilization of polyamidoamine (PAMAM) dendrimer-encapsulated gold nanoparticles (Au–PAMAM) sensing interface on a Au electrode surface, followed by sequential covalent immobilization of ethyleneamineviologen (Vio) electrochemical redox marker and AFP monoclonal antibody (mAb) on the surface of Au–PAMAM. The Au–PAMAM nanocomposite not only led to increase the electrode surface area and accelerate the electron transfer kinetics, but also it could provide a highly stable matrix for the convenient conjugation of biomolecules. Upon immunorecognition of the immobilized AFP to its antibody, the Vio peak current decreased due to the hindered electron transfer reaction on the electrode surface. Through the differential pulse voltammetry (DPV) experiments, it is found that the proposed method could detect AFP antigen at a wide linear range (0.001–45ngmL−1) and a detection limit down to 130fgmL−1. The immunosensor exhibited high specificity for AFP detection, extremely short incubation time (5min), good stability and acceptable reproducibility. Moreover, the fabricated immunosensor could accurately detect AFP concentration in human serum samples demonstrated by excellent correlations with standard ELISA immunoassay. In addition, electrochemical impedance spectroscopy technique was used as an efficient alternative detection system for AFP measurement with detection limit 0.5ngmL−1 and concentration range up to 40ngmL−1. The present protocol is shown to be quite promising for clinical screening of cancer biomarkers and point-of-care diagnostics applications.

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg·mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and ~100,000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.