Detection Of Protein Biomarkers Research Articles

Transdermal Minimally Invasive Optical Multiplex Detection of Protein Biomarkers by Nanopillars Array-Embedded Microneedles.

Biomarkers detection has become essential in medical diagnostics and early detection of life-threatening diseases. Modern-day medicine relies heavily on painful and invasive tests, with the extraction of large volumes of venous blood being the most common tool of biomarker detection. These tests are time-consuming, complex, expensive and require multiple sample manipulations and trained staff. The application of "intradermal" biosensors utilizing microneedles as minimally invasive sensing elements for capillary blood biomarkers detection has gained extensive interest in the past few years as a central point-of-care (POC) detection platform. Herein, we present a diagnosis paradigm based on vertically aligned nanopillar array-embedded microneedles sampling-and-detection elements for the direct optical detection and quantification of biomarkers in capillary blood. We present here a demonstration of the simple fabrication route for the creation of a multidetection-zone silicon nanopillar array, embedded in microneedle elements, followed by their area-selective chemical modification, toward the multiplex intradermal biomarkers detection. The utilization of the rapid and specific antibody-antigen binding, combined with the intrinsically large sensing area created by the nanopillar array, enables the simultaneous efficient ultrafast and highly sensitive intradermal capillary blood sampling and detection of protein biomarkers of clinical relevance, without requiring the extraction of blood samples for the ex vivo biomarkers analysis. Through preliminary in vitro and in vivo experiments, the direct intradermal in-skin blood extraction-free platform has demonstrated excellent sensitivity (low pM) and specificity for the accurate multiplex detection of protein biomarkers in capillary blood.

Revisiting ELISA with in situ amplification of biomarkers to boost its sensitivity

The enzyme-linked immunosorbent assay (ELISA) stands as a pivotal instrument in diagnostics and biomedical research, widely used for the detection and quantification of specific proteins associated with diseases. Despite its critical role, ELISA's ability to identify low-concentration proteins remains a significant challenge, hindering its effectiveness in early diagnostics, an area of growing importance. This study introduces a novel approach termed nucleic acid-templated amplification (NATA) to enhance ELISA's sensitivity without significantly modifying the common practices of the present ELISA. Drawing inspiration from viral proliferation mechanisms, our approach integrates a cell-free protein synthesis step using DNA sequences that encode the target biomarkers. This approach enables an in situ 'amplification' of target proteins during ELISA, boosting their concentrations to detectable levels. Our results demonstrate a substantial enhancement in sensitivity, with ELISA now being able to detect protein biomarkers at femtomolar concentrations—approaching the sensitivity range typically reserved for PCR in nucleic acid analysis. This breakthrough not only broadens ELISA’s applicability in early disease diagnosis and monitoring therapeutic interventions but also marks a significant advancement in protein biomarker detection technology. With its modular design, our method also offers high versatility and sensitivity, offering an advanced approach for protein detection in clinical diagnostics and research.

Exploring the Protein Signature of Endometrial Cancer: A Comprehensive Review Through Diverse Samples and Mass Spectrometry-Based Proteomics

Endometrial cancer (EC) is increasing incidence in among women, and it constitutes a health problem for women globally. An important aspect of EC management involves the use of protein biomarkers for early detection and monitoring. Protein biomarkers allow the identification of high-risk patients, detecting the disease in its early stages, and the assessment of treatment responses. Mass spectrometry (MS)-based proteomics offers robust analytical techniques and a comprehensive understanding of proteins. Proteomics methods allow scientists to investigate both the quantities and function of proteins. Thus, it provides valuable insights into how proteins are altered under different conditions. This review summarizes recent advances in MS-based proteomic biomarker discovery for EC, focusing on different sample types and MS-based techniques used in clinical studies. The review emphasized in detail the most commonly used key sources such as blood, urine, vaginal fluids and tissue. Furthermore, MS-based proteomics techniques such as untargeted, targeted, sequential window acquisition of all theoretical mass spectra (SWATH-MS) and mass spectrometry imaging used in discovery and validation/validation phases were evaluated. This review highlights the importance of biomarker discovery and clinical translation to improve diagnostic and therapeutic outcomes in EC. It aims to provide a comprehensive overview of MS-based proteomics in EC, guiding future research and clinical applications.

Fluorescent aptasensor based on competitive recognition strategies and point-of-care testing system for brain natriuretic peptide detection

Brain natriuretic peptide (BNP) is the preferred biomarker for clinical analysis, widely used in early screening and prognostic monitoring of cardiovascular and neurological diseases. Due to the low concentration and short half-life of BNP in the blood, its sensitive detection remains a challenge that must be overcome. With the rapid development of molecular diagnosis and point-of-care testing (POCT), developing a BNP biosensor with high sensitivity, good stability, accuracy, speed, and low-cost is of great significance for clinical applications and emergency diagnosis. However, BNP in human blood exists in various forms, and traditional fluorescence sensors may lead to overestimation of the concentration. In this work, we constructed an “on” fluorescent aptasensor based on competitive recognition strategy for quantitative detection of BNP, and built a fluorescence sensing detection system based on smartphone to achieve rapid on-site detection. We designed a specific aptamer labeled with carboxyfluorescein (FAM) with a simple structure and high biological affinity for selective capture of BNP, and utilized the large specific surface area and excellent fluorescence quenching effect of carboxylated graphene oxide (GO-COOH) as a carrier and quencher for the aptasensor. By optimizing experimental parameters such as aptamer and GO-COOH concentrations, as well as quenching/recovery time, linear sensitive detection of BNP was achieved in the concentration range of 0.01 ng/mL–10 ng/mL, with a detection limit as low as 10 pg/mL, good specificity, and excellent application potential in artificial serum spiking experiments. Additionally, a stable and sensitive fluorescence signal collection system has been developed to meet the needs of portable detection, breaking through the usage environment of traditional fluorescence detection equipment and filling the gap of portable fluorescence biosensing. In summary, we have creatively proposed a fluorescent aptasensor that enables rapid and sensitive detection of BNP and the development of low-cost fluorescent biosensors. The combination with POCT has shown broad application prospects and also provides some ideas for the fluorescence detection of other protein biomarkers.

Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions.

DNA-templated reactions have found wide applications in sensing and drug discovery. However, this strategy has been limited to the use of nucleic acids as templating elements to direct the proximity effect. Herein, we describe a versatile protein-templated split aptamer click ligation reaction (PT-SpA-CLR) in which the protein template-induced covalent proximity ligation of split aptamer elements enables translating protein/aptamer binding events into the output of ligated DNA products. A ligation yield of >80% is observed for three model protein templates, including VEGF165, PDGF-BB, and SARS-CoV-2 S1. The ligation reaction compensates for the weakness of reduced binding affinity resulting from splitting the aptamer, as evidenced by an approximately 2-fold lower dissociation constant than the non-ligated system. This newly developed PT-SpA-CLR strategy is further integrated with colorimetric or fluorescent reporting mechanisms to achieve easy-to-use and low-cost biosensors utilizing ligation to produce a fully active G-quadruplex or an RNA-cleaving DNAzyme to report protein binding. Both assays can achieve specific detection of an intended protein target with a limit of detection at the picomolar level even when challenged in biological samples. The combined PT-SpA-CLR and versatile sensing strategies offer attractive universal platforms for efficient detection of protein biomarkers.

CRISPR/Cas12a powered sandwich aptamer assay coupled with effective DNA self-assembly amplification for sensitive detection of immunoglobulin E

Sensitive and accurate detection of protein biomarkers is of significance for disease diagnosis. Recently, CRISPR/Cas system has emerged as a powerful signal amplifier in the field of sensing technology. We report a CRISPR/Cas12a powered sandwich aptamer assay (CAPSA) for protein analysis coupled with effective DNA self-assembly amplifications. The target protein is sandwiched between the capture antibody on the microplate and the aptamer detection probe. The aptamer is engineered with a specific initiator to trigger streptavidin (SA)-biotin-based dendritic amplification reaction, yielding a DNA polymer with multiple repeats of active DNA (acDNA) as Cas12a recognition sites. With the addition of Cas12a reporter system, the acDNA activates Cas12a to cleave DNA reporters, producing amplified fluorescence signals. Particularly, we also describe an innovative one-step DNA self-assembly amplification strategy to meet the demands for fast point-of-care testing, which can be directly integrated into the CAPSA without additional operation steps. The proposed methods were demonstrated by detecting immunoglobulin E (IgE), which plays a vital role in allergic reactions. Our proposed one-step DNA self-assembly amplified CAPSA enabled us to detect IgE as low as 0.6 pM, exhibiting high selectivity and satisfactory performance in complex sample analysis. Collectively, this method combines the efficient signal amplification of DNA self-assembly with the high trans-cleavage activity of Cas12a, offering a simple, universal, and double-amplified approach for sensitive analysis of protein biomarkers.

Highly sensitive electrochemiluminescence sensing platform based on copper nanoclusters synthesized via a DNA nanoribbon template for the detection of cancer cells

Herein, we presented a simple ultra-sensitive "on-off-on" electrochemiluminescence (ECL) biosensor that uses copper nanoclusters (CuNCs) synthesized via a DNA nanoribbon (DNR) template (DNR-CuNCs) as a sensing platform coupled with target-cycle amplification for the sensitive and rapid detection of cancer cells that overexpress proteins. DNR templated DNR-CuNCs, a well-ordered ECL emitter, was integrated into the nucleic acid framework structure to self-assembly, which enhanced the ECL intensity benefit from reduced band gap and accelerated electron transfer reaction of CuNCs. Additionally, an optimized exonuclease III (Exo III)-assisted cycle amplification method was introduced to efficiently convert trace target biomarkers into a substantial output of DNA, significantly improving target conversion efficiency and boosting the ECL signal. Consequently, MUC1 proteins were sensitively detected by the ECL biosensor throughout a broad concentration range, from 1 fg·mL−1 to 1 ng·mL−1, with a low limit of detection (LOD) of 0.061 fg·mL−1, and successfully detected MUC1-overexpressing cancer cells (using MCF-7 cancer cells as a model), accurately identifying MCF-7 cells in the concentration range of 10–100,000 cells·mL−1 with a LOD of 5 cells·mL−1. This research, for the first time, introduced a DNR-CuNCs ECL biosensor for the highly sensitive detection of living cells and their associated surface proteins. The detection of biomarker proteins and specific cells is greatly promising by applying this simple, sensitive, and specific strategy.

Dynamic and large field of view photonic resonator absorption microscopy for ultrasensitive digital resolution detection of nucleic acid and protein biomarkers

In this paper, we describe a biosensing instrument based on our previously developed photonic resonator absorption microscope (PRAM) that incorporates autofocus, digital representation of the gold nanoparticle (AuNP) accumulation, and the ability to gather time-series image sequences of AuNP attachment and detachment from the photonic crystal (PC) surface. The combined capabilities are used to fully automate PRAM image collection during biomolecular assays to enable tiling of PRAM images to provide millimeter-scale field of view. The instrument can also gather PRAM “movies” that enables digital showcasing and dynamic counting AuNPs as they arrive and depart from the PC surface. We utilize the capabilities in the context of two biomolecular assays for detection of protein biomarkers in a conventional AuNP-tagged sandwich format. Utilizing dynamic counting of AuNP attachment and detachment events during the assay we present a detection for microRNA-375 (miRNA-375) down to 1 aM with a 10-min, room temperature, enzyme-free approach, while revealing characteristics of the binding-rate and unbinding-rate of the biomolecular interactions. Our instrument can potentially find broad applications in multiplexed point-of-care diagnostic testing, and as a general-purpose tool for quantitative characterization of biomolecular binding kinetics with single-molecule resolution.

Lab-in-a-Vial Rapid Test for Internet of Things-Embedded Point-of-Healthcare Protein Biomarker Detection in Bodily Fluids.

Amateurs often struggle with detecting and quantifying protein biomarkers in body fluids due to the high expertise required. This study introduces a Lab-in-a-Vial (LV) rapid diagnostic platform, featuring hydrangea-like platinum nanozymes (PtNH), for rapid, accurate detection and quantification of protein biomarkers on-site within 15min. This method significantly enhances detection sensitivity for various biomarkers in body fluids, surpassing traditional methods such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays (LFA) by ≈250 to 1300 times. The LV platform uses a glass vial coated with specific bioreceptors such as antigens or antibodies, enabling rapid in vitro evaluation of disease risk from small fluid samples, similar to a personal ELISA-like point-of-care test (POCT). It overcomes challenges in on-site biomarker detection, allowing both detection and quantification through a portable wireless spectrometer for healthcare internet of things (H-IoT). The platform's effectiveness and adaptability are confirmed using IgG/IgM antibodies from SARS-CoV-2 infected patients and nuclear matrix protein (NMP22) from urothelial carcinoma (UC) patients as biomarkers. These tests demonstrated its accuracy and flexibility. This approach offers vast potential for diverse disease applications, provided that the relevant protein biomarkers in bodily fluids are identified.

Proteome Fishing for CRISPR/Cas12a-Based Orthogonal Multiplex Aptamer Sensing.

Detection of serum protein biomarkers is extremely challenging owing to the superior complexity of serum. Here, we report a method of proteome fishing from the serum. It uses a magnetic nanoparticle-protein corona and a multiplexed aptamer panel, which we incubated with the nanoparticle-protein corona for biomarker recognition. To transfer protein biomarker detection to aptamer detection, we established a CRISPR/Cas12a-based orthogonal multiplex aptamer sensing (COMPASS) platform by profiling the aptamers of protein corona with clinical nonsmall cell lung cancer (NSCLC) serum samples. Furthermore, we determined the four out of nine (FOON) panel (including HE4, NSE, AFP, and VEGF165) to be the most cost-effective and accurate panel for COMPASS in NSCLC diagnosis. The diagnostic accuracy of NSCLC by the FOON panel with internal and external cohorts was 95.56% (ROC-AUC = 99.40%) and 89.58% (ROC-AUC = 95.41%), respectively. Our developed COMPASS technology circumvents the otherwise challenging multiplexed serum protein amplification problem and avoids aptamer degradation in serum. Therefore, this novel COMPASS could lead to the development of a facile, cost-effective, intelligent, and high-throughput diagnostic platform for large-cohort cancer screening.

Comprehensive serum proteomics profiles and potential protein biomarkers for the early detection of advanced adenoma and colorectal cancer.

The majority of colorectal cancer (CRC) cases develop from precursor advanced adenoma (AA). With the development of proteomics technologies, blood protein biomarkers have potential applications in the early screening of AA and CRC in the general population. To identify serum protein biomarkers for the early screening of AA and CRC. We collected 43 serum samples from 8 normal controls (NCs), 19 AA patients and 16 CRC patients at China-Japan Friendship Hospital. Quantitative proteomic analysis was performed using liquid chromatography-mass spectrometry/mass spectrometry and data independent acquisition, and differentially expressed proteins (DEPs) with P-values < 0.05 and absolute fold changes > 1.5 were screened out, followed by bioinformatics analysis. Prognosis was further analyzed based on public databases, and proteins expression in tissues were validated by immunohistochemistry. A total of 2132 proteins and 17365 peptides were identified in the serum samples. There were 459 upregulated proteins and 118 downregulated proteins in the NC vs AA group, 289 and 180 in the NC vs CRC group, and 52 and 248 in the AA vs CRC group, respectively. Bioinformatic analysis revealed that these DEPs had different functions and participated in extensive signaling pathways. We also identified DIAPH1, VASP, RAB11B, LBP, SAR1A, TUBGCP5, and DOK3 as important proteins for the progression of AA and CRC. Furthermore, VASP (P < 0.01), LBP (P = 0.01), TUBGCP5 (P < 0.01), and DOK3 (P < 0.01) were associated with a poor prognosis. In addition, we propose that LBP and VASP may be more promising protein biomarkers for the early screening of colorectal tumors. Our study elucidated the serum proteomic profiles of AA and CRC patients, and the identified proteins, such as LBP and VASP, may contribute to the early detection of AA and CRC.

Rapid Plasma Proteome Profiling via Nanoparticle Protein Corona and Direct Infusion Mass Spectrometry.

Noninvasive detection of protein biomarkers in plasma is crucial for clinical purposes. Liquid chromatography-mass spectrometry (LC-MS) is the gold standard technique for plasma proteome analysis, but despite recent advances, it remains limited by throughput, cost, and coverage. Here, we introduce a new hybrid method that integrates direct infusion shotgun proteome analysis (DISPA) with nanoparticle (NP) protein corona enrichment for high-throughput and efficient plasma proteomic profiling. We realized over 280 protein identifications in 1.4 min collection time, which enables a potential throughput of approximately 1000 samples daily. The identified proteins are involved in valuable pathways, and 44 of the proteins are FDA-approved biomarkers. The robustness and quantitative accuracy of this method were evaluated across multiple NPs and concentrations with a mean coefficient of variation of 17%. Moreover, different protein corona profiles were observed among various NPs based on their distinct surface modifications, and all NP protein profiles exhibited deeper coverage and better quantification than neat plasma. Our streamlined workflow merges coverage and throughput with precise quantification, leveraging both DISPA and NP protein corona enrichment. This underscores the significant potential of DISPA when paired with NP sample preparation techniques for plasma proteome studies.

Quantitative SERS detection of serum protein biomarkers for assessment of tumor microwave ablation outcomes

Microwave ablation (MWA) is widely applied in the treatment of cancer, especially hepatocellular carcinoma (HCC). However, the lack of indicators for evaluating ablation completeness limits the success rate and wider application of MWA. This study therefore aims to identify novel serum biomarkers for evaluating the completeness of ablation. Multiple cytokines are preliminarily identified through cell experiments, and the potential as biomarkers is evaluated using clinical serum samples from patients with HCC before and after MWA. CCL20 and EGF are identified as promising biomarkers of the completeness of MWA. Additionally, we develop a highly sensitive and specific quantitative detection method for detecting CCL20 and EGF levels by fabricating a sandwich surface-enhanced Raman spectroscopy (SERS) nano-architecture amplification-based immunosensor. The linear detection range of this SERS platform is 0.1 pg/mL–1 ng/mL with the limit of detections (LoDs) decreasing to 0.082 pg/mL for CCL20 and 0.096 pg/mL for EGF. The remarkable consistency of the SERS immunosensor and ELISA in detecting CCL20 and EGF serum levels highlights the promising clinical utility of SERS in biomarker detection. The exceptional sensitivity of SERS in detecting CCL20 and EGF offers a potential avenue for enhancing the assessment of clinical MWA completeness, consequently leading to improved patient survival rates.

Ultra-fast detection of pathogens and protein biomarkers using a low-cost silicon plasmonic biosensing platform

In this paper, we report, for the first time to our knowledge, a new versatile and future proof diagnostic platform for diverse applications and heterogeneous biological targets. This platform relies on a plasmonic-augmented silicon photonic biochip to detect bacteria and protein biomarkers within minutes. To demonstrate the potential of this platform in diverse applications, we demonstrate the detection of two heterogeneous targets, the bacterium Escherichia coli (E. coli) and the molecule C-reactive protein (CRP) using a universal detection method. E. coli is one of the most commonly encountered bacterial pathogens involved in food monitoring, food born infections and water contamination applications, while CRP is a well-established disease severity indicator frequently used in common clinical practice. The biochip used is fully compatible with CMOS semiconductor manufacturing, while it hosts biosensors arrays for any combination of detection assays. Each biosensor exploits a 70 μm long aluminum plasmonic transducer within a silicon nitride waveguide-based Mach–Zehnder Interferometer. Each aluminum surface was silanized and biomodified with specific antibodies. Biosensing experiments revealed that CRP can be detected in diverse sample mediums, and E. coli detection was achieved in buffer at concentrations as low as 10 cells/ml within 13–25 min. The results are in good agreement with preceding numerical simulations. The modular nature of the reported biosensing platform makes it scalable and customizable, allowing nearly any combination of diagnostic tests targeting pathogens and proteins to be integrated on the same biochip for food quality monitoring, environmental monitoring, drug discovery and modern cell therapy manufacturing, or biomedical applications.

Machine Learning-Assisted High-Throughput Identification and Quantification of Protein Biomarkers with Printed Heterochains.

Advanced in vitro diagnosis technologies are highly desirable in early detection, prognosis, and progression monitoring of diseases. Here, we engineer a multiplex protein biosensing strategy based on the tunable liquid confinement self-assembly of multi-material heterochains, which show improved sensitivity, throughput, and accuracy compared to standard ELISA kits. By controlling the material combination and the number of ligand nanoparticles (NPs), we observe robust near-field enhancement as well as both strong electromagnetic resonance in polymer-semiconductor heterochains. In particular, their optical signals show a linear response to the coordination number of the semiconductor NPs in a wide range. Accordingly, a visible nanophotonic biosensor is developed by functionalizing antibodies on central polymer chains that can identify target proteins attached to semiconductor NPs. This allows for the specific detection of multiple protein biomarkers from healthy people and pancreatic cancer patients in one step with an ultralow detection limit (1 pg/mL). Furthermore, rapid and high-throughput quantification of protein expression levels in diverse clinical samples such as buffer, urine, and serum is achieved by combining a neural network algorithm, with an average accuracy of 97.3%. This work demonstrates that the heterochain-based biosensor is an exemplary candidate for constructing next-generation diagnostic tools and suitable for many clinical settings.

High-throughput light-induced immunoassay with milliwatt-level laser under one-minute optical antibody-coating on nanoparticle-imprinted substrate

The efficient detection of protein biomarkers is critical for public health. However, the sensitivity of conventional antigen test kits is relatively low for early diagnosis, and laboratory immunoassays require complex pretreatment processes overnight. If target nanomaterials could be remotely guided to the detection site, simpler and faster methods would be developed. Here, we reveal the mechanism of light-induced immunoassay that anti-spike-protein antibodies for SARS-CoV-2 were coated on our developed nanoparticle-imprinted plasmonic substrate (NPI-PS) over the submillimeter area within one minute and nanoparticles modified with spike proteins can be selectively detected within a few minutes at one or two orders of higher sensitivity via a two-step optical condensation using NPI-PS. NPI-PS exhibits high-performance optical condensation with high photothermal properties even under milliwatt-class nonresonant laser irradiation, enabling a wide range of quantitative measurements. These findings support an innovative strategy to mitigate pandemic threats and various diseases through the high-throughput detection of protein biomarkers.

Polyaromatic Hydrocarbon-Functionalized 2D MXene-Based 3D Porous Antifouling Nanocomposite with Long Shelf Life for High-Performance Electrochemical Immunosensor Applications.

Affinity-based electrochemical (AEC) biosensors have gained more attention in the field of point-of-care management. However, AEC sensing is hampered by biofouling of the electrode surface and degradation of the antifouling material. Therefore, a breakthrough in antifouling nanomaterials is crucial for the fabrication of reliable AEC biosensors. Herein, for the first time, we propose 1-pyrenebutyric acid-functionalized MXene to develop an antifouling nanocomposite to resist biofouling in the immunosensors. The nanocomposite consisted of a 3D porous network of bovine serum albumin cross-linked with glutaraldehyde with functionalized MXene as conductive nanofillers, where the inherited oxidation resistance property of functionalized MXene improved the electrochemical lifetime of the nanocomposite. On the other hand, the size-extruded porous structure of the nanocomposite inhibited the biofouling activity on the electrode surface for up to 90 days in real samples. As a proof of concept, the antifouling nanocomposite was utilized to fabricate a multiplexed immunosensor for the detection of C-reactive protein (CRP) and ferritin biomarkers. The fabricated sensor showed good selectivity over time and an excellent limit of detection for CRP and ferritin of 6.2 and 4.2 pg/mL, respectively. This research successfully demonstrated that functionalized MXene-based antifouling nanocomposites have great potential to develop high-performance and low-cost immunosensors.

Sea Anemone-Inspired Conducting Polymer Sensing Platform for Integrated Detection of Tumor Protein Marker and Circulating Tumor Cell.

Combining the detection of tumor protein markers with the capture of circulating tumor cells (CTCs) represents an ultra-promising approach for early tumor detection. However, current methodologies have not yet achieved the necessary low detection limits and efficient capture. Here, a novel polypyrrole nanotentacles sensing platform featuring anemone-like structures capable of simultaneously detecting protein biomarkers and capturing CTCs is introduced. The incorporation of nanotentacles significantly enhances the electrode surface area, providing abundant active sites for antibody binding. This enhancement allows detecting nucleus matrix protein22 and bladder tumor antigen with 2.39 and 3.12 pg mL-1 detection limit, respectively. Furthermore, the developed sensing platform effectively captures MCF-7 cells in blood samples with a detection limit of fewer than 10 cells mL-1, attributed to the synergistic multivalent binding facilitated by the specific recognition antibodies and the positive charge on the nanotentacles surface. This sensing platform demonstrates excellent detection capabilities and outstanding capture efficiency, offering a simple, accurate, and efficient strategy for early tumor detection.

Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors.

Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1% v/v mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions.

Label-free and label-based electrochemical detection of disease biomarker proteins.

Biosensors, analytical devices integrating biological sensing elements with physicochemical transducers, have gained prominence as rapid and convenient tools for monitoring human health status using biochemical analytes. Due to its cost-effectiveness, simplicity, portability, and user-friendliness, electrochemical detection has emerged as a widely adopted method in biosensor applications. Crucially, biosensors enable early disease diagnosis by detecting protein biomarkers associated with various conditions. These biomarkers offer an objective indication of medical conditions that can be accurately observed from outside the patient. This review comprehensively documents both label-free and labelled detection methods in electrochemical biosensor techniques. Label-free detection mechanisms elicit response signals upon analyte molecule binding to the sensor surface, while labelled detection employs molecular labels such as enzymes, nanoparticles, and fluorescent tags. The selection between label-free and labelled detection methods depends on various factors, including the biomolecular compound used, analyte type and biological binding site, biosensor design, sample volume, operational costs, analysis time, and desired detection limit. Focusing on the past six years, this review highlights the application of label-free and labelled electrochemical biosensors for detecting protein biomarkers of diseases.