Label-free Detection Of Biomarkers Research Articles

Silk fibroin-based wearable SERS biosensor for simultaneous sweat monitoring of creatinine and uric acid

Sweat biomarkers have the potential to offer valuable clinical insights into an individual's health and disease condition. Current sensors predominantly utilize enzymes and antibodies as biometric components to measure biomarkers present in sweat quantitatively. However, enzymes and antibodies are susceptible to interference by environmental factors, which may affect the performance of the sensor. Herein, we present a wearable microfluidic surface-enhanced Raman scattering (SERS) biosensor that enables the non-invasive and label-free detection of biomarkers in sweat. Concretely, we developed a bimetallic self-assembled anti-opal array structure with uniform hot spots, enhanced the Raman scattering effect, and integrated it into a silk fibroin-based sensing patch. Utilizing a silk fibroin substrate in the wearable SERS sensor imparts desirable properties such as softness, breathability, and biocompatibility, which enables the sensor to establish close contact with the skin without causing chemical or physical irritation. In addition, introducing microfluidic channels enables the controlled and high temporal resolution management of sweat, facilitating more efficient sweat collection. The proposed label-free SERS sensor can offer chemical ‘fingerprint’ information, enabling the identification of sweat analytes. As an illustration of the feasibility, we have effectively monitored the creatinine and uric acid levels in sweat. This study presents a versatile and highly sensitive approach for the simultaneous detection of biomarkers in human sweat, showcasing significant potential for application in point-of-care monitoring.

Modular probe integrating with quantum dots based versatile platform for sensitive and label-free biomarker detection

Multiplexed analysis of biomarkers in a single sample tube is essential for accurate diagnosis and therapy of diseases. However, the existing detection platforms suffer from many drawbacks, such as low specificity, limited applicable sceneries, and complicated operation. Hence, it is highly important to develop a versatile biomarker detection platform that can be used for disease diagnosis and pathophysiological research. In this study, we provide a versatile method for detecting biomarkers using dual-loop probes and quantum dots (QDs). This approach utilizes a dual-loop probe that consists of a recognition module for identifying specific targets, a template recognition module for initiating subsequent chain replacement cycles, and a signal module for facilitating the fixation of QDs on the 96-well plate. The lower limit of detection for miRNA-21 is determined to be at the aM level. Furthermore, this design may be easily expanded to simultaneously detect several targets, such as miRNA and C-reactive protein. The experimental results demonstrated the successful construction of the versatile biomarkers detection platform, and indicated that the sensitive and versatile platform has significant potential in the areas of bio-sensing, clinical diagnostics, and environmental sample analysis.

Multisensors based on electrolyte-gated organic field-effect transistors (EGOFETs) with aptamers as recognition elements: current state of research

The review gives a systematic account of published data devoted to multisensor technologies for manufacturing effective miniature devices capable of simultaneous accurate determination of several analytes and/or characteristics of biological fluids. The use of electrolyte-gated field-effect transistors as a biosensing platform is considered in detail. The devices based on these transistors demonstrate record-low limits of detection and are easy for mass production. The use of electrolyte-gated field-effect transistors in combination with aptamers capable of specifically binding to various analytes forms the grounds for innovations in early medical diagnosis (label-free biomarker detection). The most successful examples of multisensor devices using these transistors are presented, and the prospects of using aptamers as a recognition element in these devices are demonstrated.<br>Bibliography — 154 references.

Structural Properties of Alkanethiolates and Their Influence on The Stability and Reproducibility of Impedimetric Sensing

The utilization and commercialization of electronic based point of care (PoC) diagnostic devices has been hindered by the lack of repeatability and stability associated with such devices. Consequently, the enzyme-linked immunosorbent assay (ELISA) remains the dominant immunoassay used for the detection of serological diseases despite the numerous drawbacks associated with this platform.1, 2 Most notably, the ELISA relies on trained laboratory personnel to perform the assay in a centralized laboratory, which increases both the cost and time to actionable treatment. An electrochemical impedance biosensor is an example of a label-free biosensing platform that has high sensitivity and can be easily miniaturized and mass produced at a low cost, thus improving the time to actionable treatment.3-7 It has been shown that the repeatability and stability of electronic based biosensors is not associated with the transducing element but rather is dependent on the sensor-molecule interface.8 This interface typically consists of an alkanethiolate-based self-assembled monolayer (SAM) chemisorbed onto a gold (Au) substrate. In the case of Electrochemical Impedance Spectroscopy (EIS) sensing, the SAM forms a barrier to the ionic transport of the selected redox coupling agent. The molecular structure of the alkanethiolate determine the uniformity and density of the SAM, which directly affects its ability to impede the ionic transport of the redox coupling agent. Molecular dynamics studies have shown that shorter, less-ordered alkanethiolates rotate more easily than longer alkylthiolates.9 Their ability to rotate facilitates the gradual coverage of pinholes and defects present on the sensor-molecule interface.9 A separate study has shown that short chain (6C) alkanethiolates leads to significant drift in EIS measurements.10 The drift in charge transfer resistance of the 6C SAM was hypothesized to be associated with the surface reorganization of these thiols. The observed drift in the SAM measurements has been reported to be greater in magnitude than the measured change in charge transfer resistance upon binding of a receptor, thus compromising the reliability of biosensing measurements.10 This work uses a 16C alkanethiolate SAM to show that the stability and reproducibility of EIS measurements is directly dependent on the coverage of pinholes and defects present on the Au substrate as well as the crystallinity of the SAM. Cyclic Voltammetry (CV) and X-ray Photoelectron Spectroscopy (XPS) were used to measure the density and uniformity of the SAM, respectively. We show that stability in EIS measurements of the sensor-molecule interface directly corresponds to stable and reproducible measurements associated with the attachment of the selected receptor and lastly of the target analyte. Thus, through the rational design of a stable sensor-molecule interface we can minimize the drift associated with the SAM, and consequently the receptor interface. Thus, improving both the sensitivity and limit of detection of EIS based sensors.References Yalow, R. S.; Berson, S. A., Immunoassay of endogenous plasma insulin in man. The Journal of clinical investigation, 1960, 39 (7), 1157-1175.Prevention, C. f. D. C. a. Understanding the EIA Test. https://www.cdc.gov/lyme/diagnosistesting/labtest/twostep/eia/index.html Cui, Y., Wei, Q. Q., Park, H. K., and Lieber, C. M., Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species, Science, 2001, 293, 1289-1292.Park, I., Li, Z., Pisano, A. P., and Williams, R. S., Top-down fabricated silicon nanowire sensors for real-time chemical detection, Nanotechnology, 2001, 21, 015501.Patolsky, F., Zheng, G. F., Hayden, O., Lakadamyali, M., Zhuang, X. W., and Lieber, C. M., Electrical detection of single viruses, Proceedings of the National Academy of Sciences of the United States of America, 2004, 101, 14017-14022.Pui, T. S., Agarwal, A., Ye, F., Ton, Z. Q., Huang, Y. X., and Chen, P. Ultra-sensitive detection of adipocytokines with CMOS-compatible silicon nanowire arrays, Nanoscale, 2009, 1, 159-163.Stern, E., Vacic, A., Rajan, N. K., Criscione, J. M., Park, J., Ilic, B. R., Mooney, D. J., Reed, M. A., and Fahmy, T. M. Label-free biomarker detection from whole blood, Nature Nanotechnology, 2010, 5, 138-142.Tarasov, A.; Tsai, M.-Y.; Flynn, E. M.; Joiner, C. A.; Taylor, R. C.; Vogel, E. M., Gold-coated graphene field-effect transistors for quantitative analysis of protein–antibody interactions. 2D Mater. 2015, 2, 044008.Jiang, L., Sangeeth, C. S., Yuan, L., Thompson, D., & Nijhuis, C. A. (2015). One-Nanometer thin Monolayers remove the deleterious effect of substrate defects in Molecular Tunnel Junctions. Nano Letters, 2015 15(10), 6643-6649.Xu, X.; Makaraviciute, A.; Kumar, S.; Wen, C.; Sjödin, M.; Abdurakhmanov, E.U.; Danielson, H.; Nyholm, L.; Zhang Z.; Structural Changes of Mercaptohexanol SelfAssembled Monolayers on Gold and Their Influence on Impedimetric Aptamer Sensors, Analytical Chemistry., 2019 91 (22), 14697-14704

Microvalve-controlled miniaturized electrochemical lab-on-a-chip based biosensor for the detection of β-amyloid biomarker

Recently, miniaturized electrochemical biosensors have the advantage of real-time monitoring and label-free detection of biomarkers. However, controlled manipulation of reagent samples for specific immobilization of biomarkers remains a challenge to attend high-resolution microfluidic biosensor. Here, we designed a microfluidic channel and valves integrated electrochemical biosensor for the detection of β-amyloid (1–42) biomarker, one of the most neurotoxic peptides present in the cerebrospinal fluid, and a possible marker for the detection of Alzheimer's disease. The formation of the antibody-antigen complexes on gold microelectrode integrated inside the fluidic chambers was presented in the developed biosensor, and all the measurements are carefully controlled using pneumatic valves integrated with the device. The demonstrated microfluidic electrochemical biosensor exhibits a linear response towards various β-amyloid antigen concentrations from 2.2pM to 22μM, with an enhanced detection limit of 1.62pM. The developed technology offers an advantage of minimal reagent sample and suitable environment for miniaturized and sensitive detection of biomarkers, could also be used for the detection of other biomarkers with high efficiency.

Label-Free LSPR Prostate-Specific Antigen Immune-Sensor Based on GLAD-Fabricated Silver Nano-columns

Label-free detection of biomarkers has been recently noticed and optical biosensors showed great potential to be the method of choice in such situation. Here, we used glancing angle deposition (GLAD) method in which silver nano-columns stabilized by a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and 6-mercaptohexanol to investigate the capability of localized surface plasmon resonance (LSPR)–based silver nanochips to detect prostate-specific antigen (PSA). Using different standard solutions of PSA, limit of detection (LOD) of the nano-sensors has been calculated to be 850 pg/ml. The selectivity of the nano-sensors has also been evaluated. We showed that these nano-sensors could detect PSA in clinically acceptable sensitivity and specificity without any complicated laboratory equipment.

Detection of Biomarkers in Blood Using Liquid Crystals Assisted with Aptamer-Target Recognition Triggered in Situ Rolling Circle Amplification on Magnetic Beads.

Detection of biomarkers in body fluids is critical to both diagnosing the life-threatening diseases and optimizing therapeutic interventions. We herein report use of liquid crystals (LCs) to detect biomarkers in blood with high sensitivity and specificity by employing in situ rolling circle amplification (RCA) on magnetic beads (MBs). Specific recognition of cancer biomarkers, such as platelet derived growth factor BB (PDGF-BB) and adenosine, by aptamers leads to formation of a nucleic acid circle on MBs preassembled with ligation DNA, linear padlock DNA, and aptamers, thereby triggering in situ RCA. LCs change from dark to bright appearance after the in situ RCA products being transferred onto the LC interface decorated with octadecy trimethylammonium bromide (OTAB), which is particularly sensitive to the amplified DNA on MBs. Overall, this label-free approach takes advantages of high specificity of aptamer-based assay, efficient enrichment of signaling molecules on MBs, remarkable DNA elongation performance of the RCA reaction, and high sensitivity of LC-based assay. It successfully eliminates the matrix interference on the LC-based sensors and thus achieves at least 4 orders of magnitude improvement in sensitivity for detection of biomarkers compared to other LC-based sensors. In addition, performance of the developed sensor is comparable to that of the commercial ones. Thus, this study provides a simple, powerful, and promising approach to facilitate highly sensitive, specific, and label-free detection of biomarkers in body fluids.

Ultrahigh-Frequency, Wireless MEMS QCM Biosensor for Direct, Label-Free Detection of Biomarkers in a Large Amount of Contaminants.

Label-free biosensors, including conventional quartz-crystal-microbalance (QCM) biosensors, are seriously affected by nonspecific adsorption of contaminants involved in analyte solution, and it is exceptionally difficult to extract the sensor responses caused only by the targets. In this study, we reveal that this difficulty can be overcome with an ultrahigh-frequency, wireless QCM biosensor. The sensitivity of a QCM biosensor dramatically improves when the quartz resonator is thinned, which also makes the resonance frequency higher, causing high-speed surface movement. Contaminants weakly (nonspecifically) interact with the quartz surface, but they fail to follow the fast surface movement and cannot be detected as the loaded mass. The targets are, however, tightly captured by the receptor proteins immobilized on the surface, and they can move with the surface, contributing to the loaded mass and decreasing the resonant frequency. We have developed a MEMS QCM biosensor in which an AT-cut quartz resonator, 26 μm thick, is packaged without fixing, and we demonstrate this phenomenon by comparing the frequency changes of the fundamental (∼64 MHz) and ninth (∼576 MHz) modes. At ultrahigh-frequency operation with the ninth mode, the sensor response is independent of the amount of impurity proteins, and the binding affinity is unchanged. We then applied this method to the label-free and sandwich-free, direct detection of C-reactive protein (CRP) in serum and confirmed its applicability.

A graphene aptasensor for biomarker detection in human serum

This paper presents an affinity graphene nanosensor for detection of biomarkers in undiluted and non-desalted human serum. The affinity nanosensor is a field-effect transistor in which graphene serves as the conducting channel. The graphene surface is sequentially functionalized with a nanolayer of the polymer polyethylene glycol (PEG) and a biomarker-specific aptamer. The aptamer is able to specifically bind with and capture unlabeled biomarkers in serum. A captured biomarker induces a change in the electric conductivity of the graphene, which is measured in a buffer of optimally chosen ionic strength to determine the biomarker concentration. The PEG layer effectively rejects nonspecific adsorption of background molecules in serum while still allowing the aptamer to be readily accessible to serum-borne biomarkers and increases the effective Debye screening length on the graphene surface. Thus, the aptamer-biomarker binding sensitively changes the graphene conductivity, thereby achieving specific and label-free detection of biomarkers with high sensitivity and without the need to dilute or desalt the serum. Experimental results demonstrate that the graphene nanosensor is capable of specifically capturing human immunoglobulin E (IgE), used as a representative biomarker, in human serum in the concentration range of 50 pM–250 nM, with a resolution of 14.5 pM and a limit of detection of 47 pM.

Thin Film FETs on Flexible Substrates: a Case for Biosensing Application

Thin film transistors have been explored for biosensing applications due to their simplicity, fast reaction times and low cost. The fabrication of thin film transistors on flexible substrates open up new avenues for wearable electronic sensors for personal health monitoring. In this study, we investigate the fabrication and characterization of a Zinc Oxide based thin film transistor on a flexible substrate for biosensing application. Oxide based semiconductors have advantages such as deposition at low temperatures, better smoothness and a great potential to be used on a roll to roll fabrication for flexible applications. Zinc oxide (ZnO) serves as an ideal substrate for binding bio-recognition molecules viz., antibodies, Aptamers, enzymes, microorganisms etc. This binding is facilitated by the difference in the isoelectric points of the bio-receptor molecules (~5) and Zinc oxide (~9). Using this peculiarity, we designed back-gated bio-functionalized field effect transistors (FETs) for linker-free and label-free detection of biomarkers. The transistors were fabricated using Atomic layer deposition and sputtering techniques on a polyimide substrate. Polyimide is a stable polymer which has been used in electronic industry as a flexible substrate for various applications. The high temperature stability combined with excellent adhesion to different materials renders it a suitable substrate for this investigation. After fabrication, the transistors were characterized for their electrical parameters and material properties. The channel of the TFT was fictionalized by immobilizing antibodies that were specific to Cortisol on top of it. Cortisol is an important corticosteroid that is produced in humans by the adrenal cortex in the adrenal gland. It has been associated with maintaining healthy bodily functions and is often cited as a biomarker for quantification of stress. It is often released in response to stress and low blood glucose levels. It has been associated with several other important bodily functions and is considered as one the most important biomarkers for detecting stress. Cortisol solution was dispensed on the fictionalized FETs and the source-drain current was measured. The binding of cortisol to the immobilized antibodies causes a change in the charge transfer characteristics of the gate and thus modifies the gate voltage. This change manifests in the form of a change in drain current of the FET. Accordingly, the source-drain current is related to the change in cortisol levels of the samples exposed to the sensor.

A Wearable Electrochemical Impedance Spectroscopy Device for Detection of Glucose in Sweat Using Zinc Oxide Based Flexible Biosensors

Label-free detection of biomarkers has been of significant interest lately which proves to be a powerful means for point-of-care diagnostics. A low-power, low form factor wearable device is proposed for combinatorial detection of glucose in sweat using electrochemical impedance spectroscopy (EIS) technique. The wearable EIS device consists of a sensing analog front end and low-volume ultrasensitive flexible zinc oxide biosensors. Monoclonal antibodies specific to glucose oxidase were immobilized on a thiolated zinc oxide sensing electrode surface resulting in the modulation of charge transfer within the electrical double layer (EDL). The non-faradaic EIS response of the biosensor was used to calibrate the analog front end's response using ratiometric digital Fourier transform (DFT). In this work, thiolated biosensors are dosed with glucose concentrations ranging from 5-200 mg/dL and detection is performed using the analog front end. Continuous dose testing was performed to demonstrate the stability of sensor over prolonged periods of operation for glucose concentrations within the physiologically relevant range reported in synthetic and human sweat buffers. In addition, combinatorial detection of glucose and cortisol is performed to mimic the overall physiological response in hyperglycemic condition reported in the previous literature and demonstrate the reliability of the sensing device.

Multiplexed, label-free detection of biomarkers using aptamers and Tunable Resistive Pulse Sensing (AptaTRPS)

Diagnostics that are capable of detecting multiple biomarkers are improving the accuracy and efficiency of bioassays. In previous work we have demonstrated the potential of an aptamer-based sensor (aptasensor) utilising Tunable Resistive Pulse Sensing (TRPS). Here, we have advanced the technique identifying key experimental designs for potential POC assays. The assay utilised superparamagnetic beads, and using TRPS monitored their translocations through a pore. If the surfaces of the beads are modified with an aptamer, the frequency of beads (translocations/min) through the pore can be related to the concentration of specific proteins in the solution. Herein, we have demonstrated the successful use of TRPS to observe the binding of two proteins, to their specific aptamers simultaneously. We describe a series of experiments illustrating key factors which we believe are integral to bead-based assays and demonstrate a general method for a multiplexed assay. In summary, we have explored the effects of beads size, concentration, potential bias, pH and aptamer affinity to enhance the sensitivity and practically of a TRPS aptasensor. The method utilises the fact the binding of the aptamer to the protein results in a change in charge density on the bead surface, the isoelectric point of the protein then dominates the mobility of the beads, creating a multiplexed assay termed AptaTRPS. By alteration of the applied potential to the instrument it is possible to produce a positive signal in a simple multiplexed assay.

Multi-spot, Label-free Detection of Biomarkers in Complex Media by Reflectionless Surfaces

Abstract The measurement of the intensity of light reflected by interfaces with extremely low reflectivity in water enables the label-free, multiplex quantification of the binding between immobilized probes (e.g. antibodies) and targets in solution using extremely simple instrumental set-up. Here we show that, despite its simplicity, the method enables label-free detection in complex samples characterized by high absorbance and turbidity. Diagnostic markers of Tomato spotted wilt virus are revealed in crude plant extracts of Datura stramonium leaves with early stage infections.

Plasmon Line Shaping Using Nanocrosses for High Sensitivity Localized Surface Plasmon Resonance Sensing

The detection of small changes in the wavelength position of localized surface plasmon resonances in metal nanostructures has been used successfully in applications such as label-free detection of biomarkers. Practical implementations, however, often suffer from the large spectral width of the plasmon resonances induced by large radiative damping in the metal nanocavities. By means of a tailored design and using a reproducible nanofabrication process, high quality planar gold plasmonic nanocavities are fabricated with strongly reduced radiative damping. Moreover, additional substrate etching results in a large enhancement of the sensing volume and a subsequent increase of the sensitivity. Coherent coupling of bright and dark plasmon modes in a nanocross and nanobar is used to generate high quality factor subradiant Fano resonances. Experimental sensitivities for these modes exceeding 1000 nm/RIU with a Figure of Merit reaching 5 are demonstrated in microfluidic ensemble spectroscopy.

Label-free biomarker detection from whole blood

Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive.1-13 However, detecting these biomarkers in physiological fluid samples is difficult because of problems like biofouling and nonspecific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip captures multiple biomarkers simultaneously from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 uL sample of whole blood in less than 20 minutes. This study marks the first use of label-free nanosensors with physiologic solutions, positioning this technology for rapid translation to clinical settings.