Ultrasensitive Detection Of Protein Biomarkers Research Articles

Coupled Poly(ethylenimine) Coreactant to Enhance Electrochemiluminescence of Polymer Dots for Array Imaging of Protein Biomarkers.

Traditional electrochemiluminescent (ECL) bioanalysis suffers from the demand for excessive external coreactants and the damage of reaction intermediates. In this work, a poly(ethylenimine) (PEI)-coupled ECL emitter was proposed by covalently coupling tertiary amine-rich PEI to polymer dots (Pdots). The coupled PEI might act as a highly efficient coreactant to enhance the ECL emission of Pdots through intramolecular electron transfer, reducing the electron transfer distance between emitter and coreactant intermediates and avoiding the disadvantages of traditional ECL systems. Through modification of the PEI-Pdots with tDNA, a sequence partially complementary to cDNA that was complementary to the aptamer of target protein biomarker (aDNA), tDNA-PEI-Pdots were obtained. The biosensors were produced using Au/indium tin oxide (ITO) with an aDNA/cDNA hybrid, and an ECL imaging biosensor array was constructed for ultrasensitive detection of protein biomarkers. Using vascular endothelial growth factor 165 (VEGF165) as a protein model, the proposed ECL imaging method containing two simple incubations with target samples and then tDNA-PEI-Pdots showed a detectable range of 1 pg mL-1 to 100 ng mL-1 and a detection limit of 0.71 pg mL-1, as well as excellent performance such as low toxicity, high sensitivity, excellent selectivity, good accuracy, and acceptable fabrication reproducibility. The PEI-coupled Pdots provide a new avenue for the design of ECL emitters and the application of ECL imaging in disease biomarker detection.

Recent Advances in Electrochemiluminescence Emitters for Biosensing and Imaging of Protein Biomarkers

Electrochemiluminescence (ECL) is a light-emitting process triggered by the high energy redox between electrochemically oxidized and reduced luminophores or some coreactive intermediate radicals, representing a blooming hot topic over decades with a wide variety of bioanalytical applications. Due to the superb sensitivity, ultralow background noise, specificity, ease of integration, and real-time and in situ analysis, ECL has been developed as a convenient and versatile technique for immunodiagnostics, nucleic acid analysis, and bioimaging. Discovering highly-efficient ECL emitters has been a promising subject that will benefit the development of sensitive bioanalytical methods with prominent potential prospects. To date, the interdisciplinary integrations of electrochemistry, spectroscopy, and nanoscience have brought up the continuous emergences of novel nanomaterials which can be flexibly conjugated with specific bio-recognition elements as functional ECL emitters for bioassays. Therefore, a critical overview of recent advances in developing highly-efficient ECL emitters for ultrasensitive detection of protein biomarkers is presented in this review, where six kinds of the most promising ECL nanomaterials for biosensing and imaging of various disease-related protein biomarkers are separately introduced with references to representative works. Finally, this review discusses the ongoing opportunities and challenges of ECL emitters in developing advanced bioassays for single-molecule analysis and spatiotemporally resolved imaging of protein biomarkers with future perspectives.

Metal-Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers.

CRISPR/Cas12a shows excellent potential in disease diagnostics. However, insensitive signal conversion strategies hindered its application in detecting protein biomarkers. Here, we report a metal-organic framework (MOF)-based DNA bio-barcode integrated with the CRISPR/Cas12a system for ultrasensitive detection of protein biomarkers. In this work, zirconium-based MOF nanoparticles were comodified with antibodies and bio-barcode phosphorylated DNA as an efficient signal converter, which not only recognized the protein biomarker to form the sandwich complex but also released the bio-barcode DNA activators after MOF dissociation to activate the trans-cleavage activity of Cas12a. Due to the obvious advantages, including numerous loaded oligonucleotides, a convenient release process, and the nontoxic release reagent, this MOF-DNA bio-barcode strategy could amplify the CRISPR/Cas12a system to achieve simple and highly sensitive detection of tumor protein biomarkers with detection limits of 0.03 pg/mL (PSA) and 0.1 pg/mL (CEA), respectively. Furthermore, this platform could detect PSA directly in clinical serum samples, offering a powerful tool for early disease diagnosis.

Ultrasensitive detection of protein biomarkers by MALDI-TOF mass spectrometry based on ZnFe2O4 nanoparticles and mass tagging signal amplification

A facile MALDI-TOF mass spectrometric platform for quantitative analysis of protein biomarkers was developed based on magnetic ZnFe2O4 nanoparticles and mass tagging signal amplification. In this platform, magnetic ZnFe2O4 nanoparticles functionalized with an aptamer of the biomarker of interest was used to magnetically separate silica nanoparticles modified with another aptamer of the target biomarker and a barcoding peptide from solution phase in the presence of the biomarker of interest. After the silica nanoparticles were dissolved by KHF2, the released barcoding peptide was detected by MALDI-TOF mass spectrometry with magnetic ZnFe2O4 nanoparticles used as assisting matrix of laser desorption ionization. Since the mass spectral intensity of the barcoding peptide is directly related to the concentration of the target biomarker, the proposed platform can be applied to the quantification of the target biomarker in complex biological samples. The effectiveness of the proposed platform was tested on the detection of carcinoembryonic antigen (CEA) in serum. Experimental results revealed that the proposed platform could achieve quite reliable quantitative results for CEA in human serum samples with accuracy comparable to a commercial CEA ELISA Kit. Its limit of detection and limit of quantification for CEA were estimated to be 0.6 × 10−3 and 1.8 × 10−3 ng/mL, respectively, considerably lower than the corresponding values reported in literature. Due to its features of simplicity in design, extremely low background signal, high sensitivity and selectivity, the proposed method can be further developed to be a competitive alternative for the quantification of CEA and other protein biomarkers as well.

Direct whole blood analysis by the antigen-antibody chemically-delayed dissociation from nanosensors arrays

A wide range of fields, starting from basic research in life sciences and up to medical applications, are highly interested in the investigation and detection of biomarkers in all their forms, including proteins. However, direct analytical detection of specific protein biomarkers from a physiological biosample is still extremely challenging due to the abundant variety and amount of its components.In this work, we apply the chemically-controlled antigen-dissociation detection approach on silicon nanowires-based field-effect transistor arrays, by creating a suitable ‘chemical environment’ which enabled the clear-cut splitting of the dissociation regime window into two sub-regimes, thus allowing the complete washing of the nonspecifically adsorbed salts and biomolecules, while significantly delaying the dissociation of specific surface-bounded antigen-antibody pairs. This was accomplished by the addition of the water-miscible organic reagent ethylene glycol, which radically alters the properties of the aqueous solvent, by means of dramatically reducing its interactions with the particular protein antigen, and thus allowing for the increase in the antigen-antibody interaction strength. This in turn, deeply reduces the solubility of the surface-bound protein molecules and increases their interaction with the specific receptor antibody units, which brings to a substantial delay in the antibody-antigen dissociation behavior. This phenomenon allows the clear-cut splitting of the dissociation regime window and the quantitative and accurate analysis of proteins in physiological samples. We demonstrated the direct and quantitative detection of protein biomarkers, down to concentrations in the fM range, from unprocessed whole blood minuscule samples of only a few microliters. This work is the first demonstration on the chemically-controlled dissociation kinetics of antibody-antigen pairs by the use of water-miscible organic solvent mixtures, and its application in the direct ultrasensitive detection of protein biomarkers from whole blood samples.

DNA-Programmed plasmonic ELISA for the ultrasensitive detection of protein biomarkers.

We report a novel DNA-programmed plasmonic enzyme-linked immunosorbent assay (ELISA) for the ultrasensitive detection of protein biomarkers with the naked eye. The DNA-programmed plasmonic assay was based on two enzyme-free and isothermal nucleic acid amplification methods: hybridization chain reaction (HCR) and catalyzed hairpin assembly (CHA). In this study, a biotin-labeled DNA probe was utilized insteand of an enzyme-label probe in well-developed ELISA method. The biotin-labeled DNA probe was able to trigger the HCR and CHA processes, and the products could hybridize with DNA-modified gold nanoparticles (AuNPs) to induce the aggregation of the AuNPs and a color change in the solution. The developed method was able to detect as low as 1 pg mL-1 PSA target with the naked eye. Clinical serum samples demonstrated satisfactory results, indicating that the method is useful for early diagnostics and monitoring curative effects after a medical treatment. The developed method presents a simple and portable platform for ultrasensitive protein detection and has potential for point-of-care (POC) diagnostics in less developed areas.

Magnetic Focus Lateral Flow Sensor for Detection of Cervical Cancer Biomarkers.

We report on a magnetic focus lateral flow biosensor (mLFS) for ultrasensitive detection of protein biomarkers in a practical format. With valosin-containing protein as a target protein, we show that the developed mLFS concept could detect as low as 25 fg/mL with magnetic focus to enhance target capture efficiency to deliver a 106-fold improvement in sensitivity compared to that of conventional lateral flow (LF) systems. The conceptualized strategy utilizes a simple magnet placed beneath the three-dimensional printed LF device to concentrate the targets at the signal zone without any additional instrumentation. In addition, protein mixtures extracted from the tissue of cervical cancer patients was also utilized to validate the sensor. To investigate the effect of magnetic focus on sensitivity, surface-enhanced Raman spectroscopy and dark-field imaging was utilized to characterize the distribution and movement of Fe3O4 core-Au shell nanoprobes in a model LF strip. Our experiments show that the magnetic focus results in an increased interaction time between the magnetic probe-labeled targets and the capture antibody, yielding a higher capture efficiency, allowing for ultrasensitive detection of the target not possible before with LF. The proposed mLFS can be utilized to detect a range of trace protein biomarkers for early diagnosis and can be combined with diverse pretreatment and signal amplification steps to query complex samples.

Nanoelectronic Platform for Ultrasensitive Detection of Protein Biomarkers in Serum using DNA Amplification

Silicon nanowire field effect transistors (NWFETs) are low noise, low power, ultrasensitive biosensors that are highly amenable to integration. However, using NWFETs to achieve direct protein detection in physiological buffers such as blood serum remains difficult due to Debye screening, nonspecific binding, and stringent functionalization requirements. In this work, we performed an indirect sandwich immunoassay in serum combined with exponential DNA amplification and pH measurement by ultrasensitive NWFET sensors. Measurements of model cytokine interleukin-2 concentrations from <20 fM to >200 pM were demonstrated, surpassing the conventional NWFET urease-based readout. Our approach paves way for future development of universal, highly sensitive, miniaturized, and integrated nanoelectronic devices that can be applied to a wide variety of analytes.

Ultra-sensitive detection of protein biomarkers for diagnosis of Alzheimer's disease.

Beta amyloid peptide, tau, and phosphorylated tau are well recognized as promising biomarkers for the diagnosis of Alzheimer's disease (AD). In this work, we developed a direct, versatile, and ultrasensitive multiplex assay for the quantification of trace amounts of these protein biomarkers for AD in different types of biological fluids including cerebrospinal fluid, serum, saliva, and urine. The detection assay is based on the immunoreaction between the target proteins and their corresponding pair of antibodies followed by fluorescence labelling with a newly developed indolium-based turn-on fluorophore, namely SIM. SIM was tailor-made as a reporter to provide a high signal-to-noise ratio for the detection assay. An exceptionally low limit of detection down to the femto-molar level was achieved in this assay with minute consumption of the sample. This versatile detection assay is capable of reliably quantifying not only the target proteins simultaneously from a CSF sample in an hour but also trace amounts of protein biomarkers in saliva and urine. This assay has a high potential to serve as a practical tool for the diagnosis of AD.

Aggregated Silver Nanoparticles Based Surface-Enhanced Raman Scattering Enzyme-Linked Immunosorbent Assay for Ultrasensitive Detection of Protein Biomarkers and Small Molecules

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

A double-enhanced strip biosensor for the rapid and ultrasensitive detection of protein biomarkers.

Combining the specific molecular recognition reaction between biotin and streptavidin with a dual-class signal amplification system, containing the unique optical properties of gold nanoparticles and the catalytic reaction of horseradish peroxidase, a double-enhanced strip biosensor was designed for the first time for a visual and rapid detection of protein biomarkers with good accuracy, especially for CEA with a detection limit of 2.9 fg mL(-1).

An enhanced LSPR fiber-optic nanoprobe for ultrasensitive detection of protein biomarkers

A miniaturized, localized surface plasmon resonance (LSPR)-coupled fiber-optic (FO) nanoprobe is reported as a biosensor that is capable of label-free, sensitive detection of a cancer protein biomarker, free prostate specific antigen (f-PSA). The biosensor is based on the LSPR at the reusable dielectric-metallic hybrid interface with a robust, gold nano-disk array at the fiber end facet that is directly fabricated using EBL and metal lift-off process. The f-PSA has been detected with a mouse anti-human PSA monoclonal antibody (mAb) as a specific receptor linked with a self-assembled monolayer at the LSPR-FO facet surfaces. Experimental investigation and data analysis found near field refractive index (RI) sensitivity at ~226nm/RIU with current LSPR-FO nanoprobe, and demonstrated the lowest limit of detection (LOD) at 100fg/mL (~3fM) of f-PSA in PBS solutions. The control experimentation using 5mg/mL bovine serum albumin in PBS and nonspecific surface test shows the excellent specificity and selectivity in the detection of f-PSA in PBS. These results present important progress towards a miniaturized, multifunctional fiber-optic technology that integrates informational communication and sensing function for developing a high performance, label-free, point-of-care (POC) device.

Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.

Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid–carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L−1 for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

Tailoring of Nanotextured Zinc Oxide Thin Films for Enhanced Biosensing

ABSTRACTThis study demonstrates the development of a zinc oxide (ZnO) based microelectrode sensor for the ultra-sensitive detection of protein biomarkers. Our research focuses on utilizing a materials-based approach to achieve this objective by utilizing ZnO as part of our biosensor for (1) improved surface binding to enhance sensitivity and (2) creating a nanotextured surface for enhanced output signal response. Nanotextured ZnO thin films were integrated onto printed circuit boards using RF magnetron sputter deposition. Films sputtered with and without the presence of oxygen were examined for possible differences in biosensor efficacy. These fabrication conditions not only dictate the number of oxygen vacancies within the film but also regulate the amount of zinc and oxygen terminated ends occurring on the material surface. The correlation between the surface terminations of the nanotextured ZnO to its performance as a biosensor was evaluated using two cross-linker molecules, dithiobis succinimidyl propionate and (3-aminopropyl)triethoxysilane, that maintain different binding chemistries to ZnO. Qualitative and quantitative assessment of cross-linker binding was accomplished using fluorescent microscopy and fluorescent intensity measurements. Electrical impedance spectroscopy (EIS) was used as the transduction mechanism for detection of the well-established cardiac biomarker, troponin-T. Utilizing EIS with a functionalized immunoassay on the ZnO surface, troponin-T was detected as low as 10 fg/mL using ZnO films sputtered without oxygen. This enhanced detection of the cardiac biomarker can be directly attributed to 1) oxygen vacancies within the metal oxide film, 2) the nanotexturing of the sensing site surface, and 3) the ability to bind a significant amount of cross-linker molecules for immobilizing capture antibodies.

Metal-enhanced fluorescent detection for protein microarrays based on a silver plasmonic substrate

This paper presents an ultrasensitive fluorescent detection method through fabricating a silver microarray substrate. Silver nanoparticles (AgNPs) and Ag@Au core-shell nanoparticles with different sizes were first synthesized by a seed-mediated growth method and the metal-enhanced fluorescence of these nanoparticles on different fluorescent dyes was investigated. The results indicated that AgNPs could act as a versatile and effective metal-enhanced fluorescence material for various fluorophores, whereas the enhanced fluorescence from Ag@Au was limited only to certain fluorophores. When the AgNPs were functionalized with aptamers and fluorescent dyes, a good analytical performance for simultaneous detection of human IgE and platelet-derived growth factor-BB (PDGF-BB) could be obtained. AgNPs were not only used as detection tags but also used to fabricate the plasmonic microarray substrate to further enhance the sensitivity of fluorescent detection. As a result, a linear response to PDGF-BB concentration was obtained in the concentration range of 16 pg mL(-1) to 50 ng mL(-1), and the detection limit was 3.2 pg mL(-1). In addition, the AgNP modified plasmonic microarrays showed remarkable recovery and no significant interference from human serum when applied to 2 ng mL(-1) PDGF-BB concentration. The plasmonic microarray substrate demonstrated both high specificity and sensitivity for protein microarray detection and this novel approach has great potential for ultrasensitive detection of protein biomarkers in the bio-medical field.

Si Nanowires Forest-Based On-Chip Biomolecular Filtering, Separation and Preconcentration Devices: Nanowires Do it All

The development of efficient biomolecular separation and purification techniques is of critical importance in modern genomics, proteomics, and biosensing areas, primarily due to the fact that most biosamples are mixtures of high diversity and complexity. Most of existent techniques lack the capability to rapidly and selectively separate and concentrate specific target proteins from a complex biosample, and are difficult to integrate with lab-on-a-chip sensing devices. Here, we demonstrate the development of an on-chip all-SiNW filtering, selective separation, desalting, and preconcentration platform for the direct analysis of whole blood and other complex biosamples. The separation of required protein analytes from raw biosamples is first performed using a antibody-modified roughness-controlled SiNWs (silicon nanowires) forest of ultralarge binding surface area, followed by the release of target proteins in a controlled liquid media, and their subsequent detection by supersensitive SiNW-based FETs arrays fabricated on the same chip platform. Importantly, this is the first demonstration of an all-NWs device for the whole direct analysis of blood samples on a single chip, able to selectively collect and separate specific low abundant proteins, while easily removing unwanted blood components (proteins, cells) and achieving desalting effects, without the requirement of time-consuming centrifugation steps, the use of desalting or affinity columns. Futhermore, we have demonstrated the use of our nanowire forest-based separation device, integrated in a single platform with downstream SiNW-based sensors arrays, for the real-time ultrasensitive detection of protein biomarkers directly from blood samples. The whole ultrasensitive protein label-free analysis process can be practically performed in less than 10 min.