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Nanopores offer significant advantages in biosensing. Conventional nanopore sensors require probe modification within the pore, and there are two major obstacles: inhomogeneity of probe modification within the pore, and distortion of the detection signal due to the uncontrollable dynamics of the target within the pore. Here, we constructed a cell‐sensing analogue nanopore (CeSa‐nanopore), by coating the outer surface of the nanopore with cell membrane. The inhomogeneity of probe modification and the uncontrollable kinetics of target‐probe binding were also addressed. Specific cells are selected to prepare CeSa‐nanopore, for example, cells with high expression of angiotensin‐converting enzyme 2 (ACE2) are used to achieve the detection of SARS‐CoV‐2. When SARS‐CoV‐2 binds to CeSa‐nanopore the surface potential changes, causing a change in the ionic current, thus enabling its detection with a detection rate of 100 %. In addition, the detection of different proteins, such as follicle‐stimulating hormone (FSH), can be achieved by changing the cell membrane coating. The identification of cancer cells in ascites can also be achieved by utilizing homologous targeting between cancer cells. Importantly, the use of CeSa‐nanopores for the detection of these targets eliminates the need for pre‐processing and significantly reduces detection time. [ABSTRACT FROM AUTHOR]

Limit of detection (LoD) is a term that is used to characterize the sensitivity of an analytical method. The existing limitation of the sensitivity of analysis using modern mass spectrometry methods has been experimentally shown to be a limiting factor in the application of proteomic technologies in medicine. This article proposes a concept of a new technology that will set a new vector of development in the development of systems for solving problems of medical diagnostics and deals with theoretical and practical aspects of creating a new technology for the detection of single biomacromolecules (in particular, proteins) in biological samples. Such technology should be based on the principle of signal registration similar to that used in a Geiger counter (also known as a Geiger–Müller counter or G-M counter), a device that automatically counts the number of ionizing particles that hit it. This counter is free from probabilistic components; it registers a signal if there is at least one target molecule in the analysis chamber. Predictive medical diagnostics require technology based on methods where sensitivity allows for the detection of single marker molecules in a biological sample volume of 1–10 µL, the smallest volume of biomaterial used in laboratory diagnostics. Creation of a detector with a sensitivity of 10−18 M would allow for the detection of one molecule in 1 µL of the sample, which fundamentally makes this approach analogous to a G-M counter for solutions. To date, bioanalytical methods are limited to a sensitivity of 10−12 M (which is approximately 1 million molecules per 1 μL), which is insufficient to capture the early stages of pathological processes. [ABSTRACT FROM AUTHOR]

Biosensors are used for the specific and sensitive detection of biomolecules. In conventional approaches, the suspected target molecules are bound to selected capture molecules and successful binding is indicated by additional labelling to enable optical readout. This labelling requires additional processing steps tailored to the application. While numerous label-free interaction assays exist, they often compromise on detection characteristics. In this context, we introduce a novel diffractometric biosensor, comprising a diffractive biosensor chip and an associated optical reader assembly. This innovative system can capture an entire assay, detecting various types of molecules in a label-free manner and present the results within in a single, comprehensive image. The applicability of the biosensor is assessed for the detection of viral DNA as well as proteins directly in human plasma, investigating different antigens. In our experiments, we achieve a detection limit of 4.2 pg/mm², which is comparable to other label-free optical biosensors. The simplicity and robustness of the method make it a compelling option for advancing biosensing technologies. This work contributes to the development of an imaging diffractometric biosensor with the potential for multiple applications in molecular interaction analysis. [ABSTRACT FROM AUTHOR]

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.

Fe3O4 magnetite nanoparticles (MNPs) were prepared using the co-precipitation method at room temperature. The MNPs were modified by coating with SiO2 and APTES to enhance their efficiency for protein detection applications. The MNPs were characterized by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy with Energy Dispersive X-ray spectroscopy (SEM/EDXS), Fourier Transform Infrared (FT-IR), Thermogravimetry Analysis (TGA), X-ray diffraction (XRD), and Vibrating Sample Magnetometer (VSM). The results indicated differences in the size of particles, as around 10-40 nm and there was observed agglomeration among the sample, which, according to SEM image. Moreover, FT-IR analysis confirmed the presence of silica and NH2 coating on the magnetic nanoparticle surface. The immobilization of amine molecules (-NH2) onto the SiO2 surface is evident in TGA. The crystalline structure of the MNPs was identified as the cubic inverse spinel structure of pure-phase polycrystalline magnetite through XRD. Results from the vibrating sample magnetometer (VSM) revealed that the prepared MNPs exhibited a high magnetization saturation (Ms) of 61.31 emu/g and a coercive field strength (Hc) of 30.94 Oe (near ferromagnetism). After being surface modification with SiO2, Ms reduced to 43.24 emu/g, which further decreased to 40.17 emu/g following modification with APTES. Additionally, the Bradford assay was used to measure the percentage binding for BSA, assessing the efficiency of protein detection application. [ABSTRACT FROM AUTHOR]

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF‐Protein‐seq) is proposed, which transforms protein information into DNA sequencing readout via DNA‐tagged antibodies and labels single cells with unique cell barcodes. In a 184‐electrode DMF‐Protein‐seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low‐adsorbed hydrophobic surface and contaminants‐isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench‐top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single‐cell level. [ABSTRACT FROM AUTHOR]

This research paper explores the qualitative analysis of protein samples using the Biuret reagent through microscale methods involving ceramic tiles, grooved tiles, and Eppendorf tubes. The study highlights the advantages of minimal reagent usage, reduced waste, and enhanced safety. The Biuret test is employed to detect peptide bonds indicative of protein presence. Protein concentrations used for the identity assay ranged from 0.1% to 1.0%. Protein was detected by all three methods compared; however, the Eppendorf method detected proteins at a concentration of 0.3% and above. The ceramic and grooved tiles could detect proteins from 0.4% and above, demonstrating that the Eppendorf method was more sensitive. The microscale adaptation of this traditional test is evaluated for its efficacy, precision, and practicality in various applications, particularly in educational and resource-limited laboratory settings. [ABSTRACT FROM AUTHOR]

In this paper, we study the drift behavior of organic electrochemical transistor (OECT) biosensors in a phosphate-buffered saline (PBS) buffer solution and human serum. Theoretical and experimental methods are illustrated in this paper to understand the origin of the drift phenomenon and the mechanism of ion diffusion in the sensing layer. The drift phenomenon is explained using a first-order kinetic model of ion adsorption into the gate material and shows very good agreement with experimental data on drift in OECTs. We show that the temporal current drift can be largely mitigated using a dual-gate OECT architecture and that dual-gate-based biosensors can increase the accuracy and sensitivity of immuno-biosensors compared to a standard single-gate design. Specific binding can be detected at a relatively low limit of detection, even in human serum. [ABSTRACT FROM AUTHOR]

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full‐length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants. [ABSTRACT FROM AUTHOR]

Introduction: 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. Method: 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. Conclusion: 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.

Biosensors are used for the specific and sensitive detection of biomolecules. In conventional approaches, the suspected target molecules are bound to selected capture molecules and successful binding is indicated by additional labelling to enable optical readout. This labelling requires additional processing steps tailored to the application. While numerous label-free interaction assays exist, they often compromise on detection characteristics. In this context, we introduce a novel diffractometric biosensor, comprising a diffractive biosensor chip and an associated optical reader assembly. This innovative system can capture an entire assay, detecting various types of molecules in a label-free manner and present the results within in a single, comprehensive image. The applicability of the biosensor is assessed for the detection of viral DNA as well as proteins directly in human plasma, investigating different antigens. In our experiments, we achieve a detection limit of 4.2 pg/mm², which is comparable to other label-free optical biosensors. The simplicity and robustness of the method make it a compelling option for advancing biosensing technologies. This work contributes to the development of an imaging diffractometric biosensor with the potential for multiple applications in molecular interaction analysis.

Single-cell multi-omics technologies have revolutionized cancer research by allowing us to examine individual cells at a molecular level. Unlike traditional bulk omics approaches, which analyze populations of cells together, single-cell multi-omics enables us to uncover the heterogeneity within tumors and understand the unique molecular characteristics of different cell populations. By doing so, we can identify rare subpopulations of cells that are influential in tumor growth, metastasis, and resistance to therapy. Moreover, single-cell multi-omics analysis provides valuable insights into the immune response triggered by various therapeutic interventions, such as immune checkpoint blockade, chemotherapy, and cell therapy. It also helps us better understand the intricate tumor microenvironment and its impact on patient prognosis and response to treatment. This comprehensive review focuses on the recent advancements in single-cell multi-omics methodologies, with an emphasis on single-cell multi-omics technologies. It highlights the important role of these techniques in uncovering the complexity of tumorigenesis and its multiple applications in cancer research, as well as their equally great contributions in other areas such as immunology. Through single-cell multi-omics, we gain a deeper understanding of cancer biology and pave the way for more precise and effective therapeutic strategies. Apart from those above, this paper also aims to introduce the advancements in live cell imaging technology, the latest developments in protein detection techniques, and explore their seamless integration with single-cell multi-omics technology.

Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors―highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope―the cantilever―the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe–target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes. [ABSTRACT FROM AUTHOR]

Straightforward methods for specifically detecting and quantifying proteins are essential for both basic and applied research and notably in synthetic biology. Previously we demonstrated that the yeast mating pathway could be hijacked to detect species‐specific fungal peptide pheromones using their corresponding mating GPCRs. Here we asked if our yeast biosensor could detect proteins in addition to peptides – a question not previously resolved in the literature. As such, we repurposed the Saccharomyces cerevisiae fungal mating pheromone α‐factor as a peptide tag and fused it terminally and internally to the protein Smt3. Our biosensor was able to detect the tagged protein in the nanomolar range using fluorescence as a read‐out. We extended the assay to four additional orthogonal peptide pheromone tags, demonstrating a cheap, non‐labor‐intensive, and high‐throughput assay compatible with multiplexing for protein detection. With its ability to detect proteins our living yeast biosensor could be useful for the optimization of protein producing cell‐factories, for building logic gates and myriad other applications in synthetic biology. [ABSTRACT FROM AUTHOR]

Due to their distinctive optical potential, controlled plasmonic features, control over size and shape, and ability to manipulate their localized surface plasmon resonance (LSPR) and their response to their surroundings, silver nanoparticle attracts special attention in the fields of the biosensor. Silver (Ag) is an appealing tool in many domains, including diagnosis, medicine delivery, environment, electronics, and as an antibacterial agent. Ag nanoparticle applications are ubiquitous today in the fields of biosensors. Numerous studies have demonstrated the effectiveness of using Ag nanoparticles to improve the specific detection of clinical indicators. The development of nanotechnology has greatly benefited the field of biosensors since nanomaterials have a high surface-to-volume ratio that enables them to operate with greater sensitivity and effectiveness. Utilizing interdisciplinary study in biology, chemistry, and material science, recent advancements in nanotechnology encouraged the manufacture of nanomaterials in a variety of shapes and sizes. In the context of disease diagnosis, this review highlighted the use of Ag nanoparticles in biosensors to detect small molecules to bigger entire cells. This paper also discusses how to make a glucose biosensor more sensitive. It describes how the detection performance of biomolecules including DNA, protein, tiny molecules, and complete cells can be improved. For future applications in the field of diagnosis, this review helps in developing an overall understanding of the clinical importance of Ag nanoparticles. These biosensors can be implemented in the field of diagnosis to speed up the response time. [ABSTRACT FROM AUTHOR]

Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay's accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses. [ABSTRACT FROM AUTHOR]

[Display omitted] Au coated magnetic polyphosphazene (MPCTP) composite particles (MPCTP@Au) were fabricated with sensitive SERS activity. The MPCTP particles were generated by coating polyphosphazene on Fe 3 O 4 nanoparticles through precipitation polycondensation of hexachlorocyclotriphosphazene and phloroglucinol. MPCTP@Au composite particles were obtained by deposition of Au nanoparticles on MPCTP by the reduction of HAuCl 4. The size and the thickness of the Au shell can be controlled by varying the amount of HAuCl 4. The magnetic core endowed the composite particles with good magnetic responsiveness, which allowed the analyte to be enriched and separated from the complex matrix, and significantly simplifying the sample pretreatment procedure. The SERS activity of MPCTP@Au composite particles were evaluated by DTNB as model Raman reporter, and the limits of detection (LOD) of DTNB was 10-8 mol/L. A high efficient SERS immunoassay system based on the MPCTP@Au substrates for the detection of immunoproteins was developed. Human IgG and rabbit IgG were quantitatively determinated simultaneously by this immunoassay system. The quantitative determination of the immunoglobulin G (IgG) was achieved and the LOD of human IgG, rabbit IgG and the mixture of human IgG and rabbit IgG were as low as 10 fg/mL, 100 pg/mL and 1 ng/mL, respectively. The results showed that the MPCTP@Au composite particles have broad application prospects as high performance SERS active substrates for immunoprotein analysis. [ABSTRACT FROM AUTHOR]

Biomolecule detection has become important in many applications such as medical diagnosis, forensic analysis, basic biological studies, and food quality assessment. In particular, the Mid-infrared range offers an important opportunity for biomolecular sensing as it covers the molecular vibrational spectra of vital biochemicals such as Deoxyribonucleic acid, Ribonucleic Acid, and proteins. In this study, a double band absorbing plasmonic nanoantenna array with two gold disk resonators is proposed. The biosensing ability of this structure was investigated using the protein-goat anti-mouse immunoglobulin G model and Polymethyl methacrylate film. The basic structural bonds of protein monolayer, namely Amide-I, Amide-II, and Amide-III showed vibrational signatures at 6010 nm (∼1664 cm−1), 6496 nm (∼1539 cm−1), and 6989 nm (∼1431 cm−1) wavelengths, respectively. In addition, the spectral response of the proposed antenna structure was investigated using a Polymethyl methacrylate film by detecting the C=O and the C-H bonds. The strong dipole moment at C=O showed a strong absorption deep at 5782 nm (∼1730 cm−1) while the C-H bond has shown a relatively low absorption deep at 3350 nm (∼2985 cm−1) and 3395 nm (∼2946 cm−1). Our findings indicate that the double spacer disk configuration detects the spectral signature of the protein monolayer and Polymethyl methacrylate film in each band, simultaneously. The dual-band can be tuned independently by carefully engineering the radii of the double disks without making an effect on the other band. The proposed structure can be used as a characterization tool for identifying unknown complex molecules by simply detecting their spectral fingerprints in each mode of the dual-band, independently. Also, this design strategy can be insight to multi-mode SEIRA platforms, where more complex chemical molecules are needed to be detected or identified in biology, chemistry, and defense areas. [ABSTRACT FROM AUTHOR]

High-sensitivity biomedical sensors could make it possible to detect and classify chemical and biological species in a variety of applications, from disease diagnosis to medication discovery, thus, boosting the likelihood of life-saving intervention. Synthesized nanowires have already produced advancements in a variety of sectors, including biological sensors over the last decade. When compared to macro-sized materials, the nanowires' large surface area-to-volume ratio increased sensitivity. Their applications for biomarker, viral, and DNA detection, as well as drug discovery, are also discussed. Self-powering, reusability, sensitivity in high ionic strength solvents, and long-term stability are all examples of recent developments. Shortly, the nanowire is likely to lead to major improvements in biomedical sensors. This review provides a full overview of the nanowire sensor's working principle and production procedure. We have discussed how to produce nanowires that can be utilized as biosensors for different bacteria and pathogens, protease, DNA and RNA, neurotransmitters, and chemical compounds. Biosensing technology has dramatically improved because of the introduction of nanowires in biosensors. This is a result of the application of new biorecognition components and transducers, improvements in the manufacture, design, and miniaturization of nanostructured devices at the micron scale, and unique approaches for the synthesis of nanowires. The versatility, robustness, and dynamic nature of sensing technologies have all improved thanks to the usage of nanowires. [ABSTRACT FROM AUTHOR]

Development of compact and low-cost electronic circuits in the biosensor field is an essential demand. Especially, the design of noise reduction sections plays an important role in the measurement of small biosensing signals. This study develops an electrical impedance measurement circuit board to apply in the protein pre-concentration and detection microfluidic chips with integrated two-electrode impedance micro-sensor. A lock-in amplifier is applied in the circuit board to measure the impedance profile in the frequency range 10–200 kHz. An investigation on several ratios of signal to noise (SNRs) is conducted to indicate the ability of the proposed design in eliminating noise signals. Subsequently, a total impedance and series capacitance-based analysis approach from the recorded impedance data is demonstrated to be suitable for this protein chip in the frequencies range from 10 to 200 kHz. The obtained results enable a potential method in point-of-care diagnostic applications. [ABSTRACT FROM AUTHOR]

The protein contents of hydrolyzed sludge supernatant are commonly determined with the Kjeldahl method, but this method suffers from complicated operations, long process times, and large quantities of chemicals consumed. In this paper, the Lowry, bicinchoninic acid (BCA), and Bradford methods were used to test the precision and spiked recovery of proteins from sludge supernatants hydrolyzed by alkaline-thermal hydrolysis (ATH), enzymatic hydrolysis (EH), and ultrasound-assisted enzymatic hydrolysis (UEH), and the results were compared with those obtained with the Kjeldahl method. For all the hydrolytic processes, the sludge protein values determined with the three tested methods were within 0.05 of each other, which met the experimental requirement for accuracy. Both the Lowry and BCA methods had recovery rates of 95–105%, while the Bradford method showed large deviations and was not highly reliable. The three protein determination methods showed significant differences with the Kjeldahl method (P<0.05). However, the relative deviation between the Kjeldahl and BCA methods was the smallest (3–5%), followed by those between the Kjeldahl and the Lowry (11–21%) and Bradford methods (21–90%), and the causes of the deviations were analyzed based on the protein hydrolysate components and the mechanisms for the different detection methods. On the basis of these results, the BCA method was chosen as the most appropriate quantification method for use with sludge protein extraction, and it was used to analyze the protein contents extracted from residual sludge samples obtained from two sewage treatment plants. The reliability of the method was verified, and this lays a foundation for the extraction and reclamation of sludge proteins. [ABSTRACT FROM AUTHOR]

Gluten consumption can lead to severe health conditions in certain individuals, and following a strict gluten-free diet is often the only effective treatment option. Therefore, it is crucial to develop a gluten detection method that is accurate, sensitive, and specific to ensure the absence of gluten. An important aspect of developing effective gluten detection tests is the implementation of an efficient gluten extraction method. In this study, we conducted an evaluation of various buffer conditions for gliadin extraction from both heat-treated and non-heat-treated food samples. These buffer conditions included ethanol, 2-mercaptoethanol, guanidine hydrochloride, detergents, chelating agents, and deep eutectic solvents. Among the tested conditions, a combination of 2-mercaptoethanol and guanidine hydrochloride demonstrated significantly higher extraction efficacy compared to most other conditions. Furthermore, we explored the use of a less toxic extraction buffer, choline chloride, which exhibited a 1.4-fold higher extraction efficiency than the combination of 2-mercaptoethanol and guanidine hydrochloride (p

In this paper, we study the drift behavior of organic electrochemical transistor (OECT) biosensors in a phosphate-buffered saline (PBS) buffer solution and human serum. Theoretical and experimental methods are illustrated in this paper to understand the origin of the drift phenomenon and the mechanism of ion diffusion in the sensing layer. The drift phenomenon is explained using a first-order kinetic model of ion adsorption into the gate material and shows very good agreement with experimental data on drift in OECTs. We show that the temporal current drift can be largely mitigated using a dual-gate OECT architecture and that dual-gate-based biosensors can increase the accuracy and sensitivity of immuno-biosensors compared to a standard single-gate design. Specific binding can be detected at a relatively low limit of detection, even in human serum.

Saliva-based biosensors are emerging as viable tools for non-invasive, painless, and easily administered household medical diagnostics. Despite their potential, the complexity of saliva, containing various non-target molecules and contaminants, presents significant challenges due to nonspecific interactions with biosensors. This study utilizes surface-charge modulated graphene field-effect transistors (SCM-GFETs) for the detection of C-reactive protein (CRP) in artificial saliva, using portable measurement apparatus. Our findings indicate that SCM-GFETs exhibit nonspecific responses to saliva components. To address this issue, a two-step preprocessing method is implemented: diluting the saliva in phosphate-buffered saline at a 1 : 104 ratio and subsequent membrane filtration. This process significantly reduced nonspecific interactions, enabling CRP detection in saliva samples. These advancements hold promise for enhancing graphene field-effect transistor technology in point-of-care diagnostic devices.

Biosensing systems are powerful biotechnological tools which are widely applied in medical and environmental settings. Herein, we provide an overview of a recently developed optical biosensing system based on the quenching abilities of graphene oxide and fluorescent bioprobes. This biosensing platform has been demonstrated to be a fast, cost-effective and reliable nanophotonic technology. In particular, it has been exploited to detect relevant analytes in real matrixes, including prostate specific E. coli and COVID-19 antibodies. Besides, this technology enabled the detection of sialidase in clinical samples to determine bacterial vaginosis. This biosensing system has recently been used to determine relevant information on the kinetics of proteins involved in the biorecognition process, everything performed in real-time and in a single step.

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb. • Capillary electrophoresis can be used to develop a standard curve to quantitatively measure protein. • Intracellular recombinant protein can be quantitatively measured with capillary electrophoresis. • There are observable differences in the intracellular titer between high- and low-production CHO clone. • Intracellular and volumetric titers are correlated between high- and low-production CHO clones. [ABSTRACT FROM AUTHOR]

The accurate detection of biological substances such as proteins has always been a hot topic in scientific research. Biomimetic sensors seek to imitate sensitive and selective mechanisms of biological systems and integrate these traits into applicable sensing platforms. Molecular imprinting technology has been extensively practiced in many domains, where it can produce various molecular recognition materials with specific recognition capabilities. Molecularly imprinted polymers (MIPs), dubbed plastic antibodies, are artificial receptors with high-affinity binding sites for a particular molecule or compound. MIPs for protein recognition are expected to have high affinity via numerous interactions between polymer matrices and multiple functional groups of the target protein. This critical review briefly describes recent advances in the synthesis, characterization, and application of MIP-based sensor platforms used to detect proteins. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8‐naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells. [ABSTRACT FROM AUTHOR]

Heterogeneous assays, such as enzyme-linked immunosorbent assays, have become indispensable for in vitro diagnostics. However, the simple, sensitive, and accurate detection is limited by their multiple washing and incubation steps, and limited amplification methods. In this study, we design a novel approach utilizing analyte-induced hindrance within the rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for simple and highly sensitive homogenous protein detection. Streptavidin (SA) and digoxin antibody (anti-Dig) are employed as representative detection models. The specific recognition of target proteins using primers modified with small molecules hinders the RCA process, preventing the activation of Cas12a′s trans -cleavage activity, thereby leading to a reduction in fluorescence intensity. Our developed platform exhibites exceptional detection performance characterized by high sensitivity, robust specificity, and significant potential for application in complex samples. By expanding the recognition elements, this platform can evolve into a versatile clinical diagnostic tool with universal applicability. In addition, this platform provides a novel direction for quantifying ultralow-concentration disease biomarkers in clinical practice. [Display omitted] • Analyte-induced hindrance within RCA-assisted CRISPR/Cas12a system for protein detection. • This platform exhibits high sensitivity, and robust specificity. • This method displays significant potential for application in complex samples. • This platform can evolve into a versatile clinical diagnostic tool with universal applicability. [ABSTRACT FROM AUTHOR]

Periodontitis is one of the most prevalent oral diseases, and the diagnosis and monitoring of periodontitis rely on clinical and imaging examinations. Matrix metalloproteinase-8 (MMP-8) in saliva is a reliable indicator of periodontitis. The traditional detection method is enzyme-linked immunosorbent assay (ELISA), which requires professional expertise and is not sensitive enough to low-abundance protein detection. In this study, we developed a nanoimmunoassay combined with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas)12a for ultrasensitive detection of MMP-8 in saliva. Antibody-DNA-gold nanoparticles (AuNPs) and trans-cleavage activity of CRISPR/Cas12a were employed for signal amplification. With this protocol, the limit of detection (LoD) for MMP-8 was reduced to the fg/mL, indicating 100-fold higher sensitivity than that of the commercial ELISA kit and its amenability for ultrasensitive detection of trace proteins. The diagnostic sensitivity of the assay was 100 %, and the specificity was 83.3 % based on the testing of 23 clinical saliva samples. The sensitivity and specificity were considerably higher than those for ELISA. Thus, this immuno-CRISPR/Cas12a assay holds promise for both diagnosing and monitoring periodontitis in clinical settings. [Display omitted] • The assay combines the AuNPs with Cas12a for signal amplification, enabling sensitive detection of MMP-8 in saliva. • This assay enables to detect the MMP-8 at fg/mL level, which is 100-fold higher sensitivity than that of ELISA method. • The immuno-CRISPR/Cas12a assay has the potential to be applied for POCT. [ABSTRACT FROM AUTHOR]

In this research, hafnium oxide (HfO 2) was first fabricated in an in-house-developed high-power pulsed magnetron sputtering system (HiPIMS) functionalized for application as a high-performance extended gate field-effect transistor (EGFET) for the detection of pH and proteins associated with Parkinson's disease (PD). PD is the second most common neurodegenerative disease in elderly people after Alzheimer's disease. The α-synuclein protein in patients' blood may be a potential biomarker in the early stage of PD. Detection of α-synuclein with high sensitivity and nonspecific binding through HfO 2 -EGFET biosensors has the advantages of low cost and simple fabrication from disposable EGs. Among the duty cycle conditions tested, HfO 2 deposited at a 25% duty cycle in HiPIMS exhibited superior pH sensitivity and linearity, with values of 56.4 mV/pH and 99.7%, respectively. The hysteresis width and drift coefficient are −3 mV and 0.3 mV/h, respectively. To overcome the Debye length limitation, 100-fold diluted phosphate buffer solution and the PMOS configuration were used to determine the α-synuclein for which the isoelectric point was 4.67. The negative gate bias in the PMOS configuration successfully causes α-synuclein to approach the surface of HfO 2 EG to achieve a sensitivity of 12.1 mV/dec and a linearity of 99.5% in the range of 0.1–1000 pg / mL. The limit of detection is 0.198 pg / mL , and the immobilized surface ultimately binds specifically, resulting in low interference signals from other related biomarkers. This promising study shows that detecting various proteins involved in different diseases and performing clinical tests can be accomplished in the future. • A high sensing performance HfO 2 extended gate prepared by HiPIMS. • Duty cycle of 25% in HiPIMS has the superior behavior. • With a proper surface treatment, α-synuclein proteins are immobilized on HfO 2. • With PMOS configuration, sensitivity of 12.1 mV/dec is obtained. [ABSTRACT FROM AUTHOR]

Aquaporin-1 (AQP1) protein plays a crucial role in intracellular and extracellular water homeostasis and fluid transport in organs and tissues associated with diverse life activities and is extremely abundant in the kidney. Accurate detection of AQP1 in urine can be applied as screening of early-stage disease. Application of magnetic preconcentration and probe-based signal amplification strategy coupling to inductively coupled plasma mass spectrometry (ICP-MS) is a more accurate, sensitive and specific detection method for AQP1 in complex biological samples compared to conventional methods. We described an element-labelling strategy based on magnetic preconcentration and probe-based immunoassay coupling to ICP-MS detection. The magnetic beads (MBs) modified with epoxy groups were capable of enriching AQP1 proteins and separating them from complex matrices. The probe constructed by conjugating anti-AQP1 antibody molecules on the surface of gold nanoparticles could specifically recognize AQP1 proteins attached on MBs and be analyzed by ICP-MS. The concentration of AQP1 protein could be precisely quantified and amplified by 14,000 times through the corresponding signal of Au atoms. This assay for AQP1 protein quantification achieved a detection limit down to 0.023 ng mL−1, a broad linear calibration curve between 0.3 ng mL−1 and 30 ng mL−1, as well as outstanding specificity. The proposed method was successfully applied to detect AQP1 protein in human urine samples, showing the potential for its applications concerning accurate AQP1 quantification. It can also screen a wide range of proteins provided the antibodies specific to these target proteins are available. [Display omitted] • AuNPs-based probe is endowed with signal amplification of Au element for target protein detection. • The method is integrated with magnetic preconcentration, probe-involved immunoassay, and ICP-MS analysis. • Magnetic preconcentration and AuNPs-labelling amplification allow for sensitive detection of AQP1 protein in urine samples. • The detection method is available for most proteins which have one antibody, not requiring antibody pairs. [ABSTRACT FROM AUTHOR]

Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors―highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope―the cantilever―the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe–target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes.

Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay’s accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses.

During past decades, enormous progress in understanding the mechanisms of the intermolecular interactions between the protein and surface at the single-molecule level has been achieved. These advances could only be possible by the ongoing development of highly sophisticated experimental methods such as atomic force microscopy, optical microscopy, surface plasmon resonance, ellipsometry, quartz crystal microbalance, conventional mass spectrometry, and, more recently, the nanomechanical systems. Here, we highlight the main findings of recent studies on the label-free single-molecule (protein) detection by nanomechanical systems including those focusing on the protein adsorption on various substrate surfaces. Since the nanomechanical techniques are capable of detecting and manipulating proteins even at the single-molecule level, therefore, they are expected to open a new way of studying the dynamics of protein functions. It is noteworthy that, in contrast to other experimental methods, where only given protein properties like molecular weight or protein stiffness can be determined, the nanomechanical systems enable a real-time measurement of the multiple protein properties (e.g., mass, stiffness, and/or generated surface stress), making them suitable for the study of protein adsorption mechanisms. Moreover, we also discuss the possible future trends in label-free detection and analysis of dynamics of protein complexes with these nanomechanical systems.

The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element". The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low molecular weight haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform's signaling mechanism to aid its design and optimization; and (3) demonstrate the analytical performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of molecular targets amenable to this rapid and convenient sensing platform.

Proteins play an important role in organisms. The fast and high-accuracy detection of proteins is demanded in various fields, such as healthcare, food safty, and biosecurity, especially in the background of the globally raging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Optical fiber sensors have great potential for protein detection due to the excellent characteristics of high sensitivity, miniaturization, and capability for remote monitoring. Over the past decades, a large number of structures have been investigated and proposed. This paper begins with an overview of different fiber sensing structures for protein detection according to the fundamental sensing mechanisms. The overview is classified into four sections, including intensity-modulation, phase-modulation, scattering, and fluorescence. In each section, we reviewed the recent advances of fiber protein sensors and compared their performance, such as sensitivity and limit of detection. And then we analyzed the advantages and disadvantages of the four kinds of biosensors. Finally, the paper concludes with the challenges faced and possible future development of optical fiber protein biosensors for further study. [ABSTRACT FROM AUTHOR]

Significant advancement has occurred in the detection methods of solution-based analytes. High-pressure liquid chromatography, gas chromatography, and other systems used for analyses are quite expensive. Therefore, there is a need for new methods and for the visible detection of analytes. Here, we demonstrate that 3-aminopropyl triethoxysilane (APTES) could impact the stability, optical, and morphology of gold nanoparticles (AuNps) in a colloidal solution. These impacts can be used to create a sensitive visual detection system. The strong impact of the APTES concentration on the ultraviolet–visible absorption spectra of the solutions is illustrated, which displays systematic and extensive red shifts. The presence of denatured proteins within a therapeutic drug product can induce a series of adverse effects. This report describes a fast, low cost, sensitive, and user-friendly platform where the plasmonic nanoparticles create visual biosensing of denatured proteins. Artificially heat stressed ferritin, glutathione, and insulin coupled to AuNps are exposed to ATES and upon denaturation of the protein or peptide, systematic blue or red shifts are observed in the absorbance spectra of the AuNps/biomolecules, and aminosilane solution. This serves as a proof-of-concept for a fast in-solution detection method for heat-stressed proteins or peptides. [ABSTRACT FROM AUTHOR]

Nanopores offer significant advantages in biosensing. Conventional nanopore sensors require probe modification within the pore, and there are two major obstacles: inhomogeneity of probe modification within the pore, and distortion of the detection signal due to the uncontrollable dynamics of the target within the pore. Here, we constructed a cell-sensing analogue nanopore (CeSa-nanopore), by coating the outer surface of the nanopore with cell membrane. The inhomogeneity of probe modification and the uncontrollable kinetics of target-probe binding were also addressed. Specific cells are selected to prepare CeSa-nanopore, for example, cells with high expression of angiotensin-converting enzyme 2 (ACE2) are used to achieve the detection of SARS-CoV-2. When SARS-CoV-2 binds to CeSa-nanopore the surface potential changes, causing a change in the ionic current, thus enabling its detection with a detection rate of 100 %. In addition, the detection of different proteins, such as follicle-stimulating hormone (FSH), can be achieved by changing the cell membrane coating. The identification of cancer cells in ascites can also be achieved by utilizing homologous targeting between cancer cells. Importantly, the use of CeSa-nanopores for the detection of these targets eliminates the need for pre-processing and significantly reduces detection time., (© 2024 Wiley-VCH GmbH.)

Thermotropic nematic liquid crystals (LC) have been utilized to sense/detect various analytes such as polymers, surfactants, lipids, etc. However, their use for protein detection depends on pre-adsorbed molecules, co-nematogens, or biomolecular agents for specificity. This approach impedes the platform's sensitivity with a detection limit for the folded proteins generally reported in the micromolar concentration range. Here, this work provides fundamental insights into the type of molecular interactions and their modulation that can drive ultrasensitive protein detection at an LC microdroplet/aqueous interface formed without adding an auxiliary co-nematogen. Using ultraviolet (UV) light treated 4-cyano-4'-pentylbiphenyl (5CB) LC and a flow-focused microfluidic device, we prepared different populations of monodisperse and highly negatively charged microdroplets in water. Adding an aqueous solution of various model proteins (α-synuclein, α-chymotrypsin, myoglobin, or bovine serum albumin, BSA) with different secondary structures and surface charges triggers a rapid radial- to bipolar-defect transition in these microdroplets. Isothermal titration calorimetry measurement and molecular dynamic simulation studies attribute this to the dominant electrostatic force-mediated adsorption of proteins at the LC/aqueous interface. Further, bioconjugation-based variation of protein surface charge allows tuning their detection limit. These findings can provide crucial physical cues for designing responsive LC systems and establishing a foundation for developing versatile, molecularly tailored, and highly specific biomolecular detection platforms., (© 2024 Wiley‐VCH GmbH.)

Abstract The ultrasensitive measurement of protein markers plays a pivotal role in the early diagnosis of infectious and progressive diseases. Recently, digital methods such as those enabled by the Simoa Planar Array technology (SP‐X System) have made significant progress in reaching ultrasensitive detection with clinically relevant protein biomarkers. The elicited Simoa technology is based on printing high‐density capturing antibodies layers on the bottom of the wells of a microtiter plate, followed by a standard sandwich‐type immunometric chemiluminescent detection. Such assay, reaching limit‐of‐detections (LODs) in the low femtomolar range, can be conveniently customized. An optimized Simoa SP‐X assay for detecting and quantifying immunoglobulin M (IgM, non‐specific indicator of inflammation) is developed herein and optimized. A full factorial experimental design is undertaken to optimize the assay, leading to a reduced experimental effort and increased quality of the information obtained concerning the traditional one‐variable‐at‐a‐time approach. The optimization process leads to an IgM LOD of 4 fm that compares well with those achieved with commercially available Simoa Planar Array kits. Remarkably, depositing both the capturing and detecting layer from a solution (0.1 µg mL−1) one order of magnitude less concentrated than in standard kits is needed, and the assay's cost is sizably reduced.

Proteins and antibodies are key biomarkers for diagnosing and monitoring specific medical conditions. Currently, gold standard techniques used for their quantification require laborious multi‐step procedures, involving high costs and slow response times. It is possible to overcome these limitations by exploiting the chemistry and programmability of DNA to design a reagentless electrochemical sensing platform. Specifically, three DNA single strands are engineered that can self‐assemble into a Y‐shaped DNA nanostructure that resembles one of the IgGs. In order to convert this DNA nanostructure into a responsive DNA‐scaffold bioreceptor, it is modified including two recognition elements, two redox tag molecules, and a thiol group. In the absence of the target, the scaffold receptor can efficiently collide with the electrode surface and generate a strong electrochemical signal. The presence of the target induces its bivalent binding, which produces steric hindrance interactions that limit the receptor's collisional activity. In its bound state, the redox tags can therefore approach the surface at a slower rate, leading to a signal decrease that is quantitatively related to the target concentration. The Y‐shape DNA scaffold sensor can detect nanomolar concentrations of antibodies and proteins in <15 min with a single‐step procedure directly in untreated biological fluids. [ABSTRACT FROM AUTHOR]

In highly sensitive bioanalytical systems intended for the detection of protein biomarkers at low and ultra-low concentrations, the efficiency of capturing target biomolecules from the volume of the analyzed sample onto the sensitive surface of the detection system is a crucial factor. Herein, the application of excess electric charge for the enhancement of transport of target biomolecules towards the sensitive surface of a detection system is considered. In our experiments, we demonstrate that an uncompensated electric charge is induced in droplets of protein-free water owing to the separation of charge in a part of the Kelvin dropper either with or without the use of an external electric field. The distribution of an excess electric charge within a protein-free water droplet is calculated. It is proposed that the efficiency of protein capturing onto the sensitive surface correlates with the sign and the amount of charge induced per every single protein biomolecule. The effect described herein can allow one to make the protein capturing controllable, enhancing the protein capturing in the desired (though small) sensitive area of a detector. This can be very useful in novel systems intended for highly sensitive detection of proteins at ultra-low (≤10−15 M) concentrations. [ABSTRACT FROM AUTHOR]

Biomarkers of disease, especially protein, show great potential for diagnosis and prognosis. For detecting a certain protein, a binding assay implementing antibodies is commonly performed. However, antibodies are not thermally stable and may cause false-positive when the sample composition is complicated. In recent years, a functional nucleic acid named aptamer has been used in many biochemical analysis cases, which is commonly selected from random sequence libraries by using the systematic evolution of ligands by exponential enrichment (SELEX) techniques. Compared to antibodies, the aptamer is more thermal stable, easier to be modified, conjugated, and amplified. Herein, an Aptamer-Based Cell-free Detection (ABCD) system was proposed to detect target protein, using epithelial cell adhesion molecule (EpCAM) as an example. We combined the robustness of aptamer in binding specificity with the signal amplification ability of CRISPR-Cas12a′s trans-cleavage activity in the ABCD system. We also demonstrated that the ABCD system could work well to detect target protein in a relatively low limit of detection (50–100 nM), which lay a foundation for the development of portable detection devices. This work highlights the superiority of the ABCD system in detecting target protein with low abundance and offers new enlightenment for future design and development.

We present a sensitive and quantitative protein detection assay that can efficiently distinguish between specific and nonspecific target binding. Our technique combines dual affinity reagents with surface-enhanced Raman spectroscopy (SERS) and chemometric analysis. We link one Raman reporter-tagged affinity reagent to gold nanoparticles and another to a gold film, such that protein-binding events create a "hot spot" with strong SERS spectra from both Raman reporter molecules. Any signal generated in this context is indicative of recognition by both affinity labels, whereas signals generated by nonspecific binding lack one or the other label, enabling us to efficiently distinguish true from false positives. We show that the number of hot spots per unit area of our substrate offers a quantitative measure of analyte concentration and demonstrate that this dual-label, SERS-linked aptasensor assay can sensitively and selectively detect human α-thrombin in 1% human serum with a limit of detection of 86 pM.

Surface engineering of nanoparticles has recently emerged as a promising technique for synthetic molecular recognition of biological analytes. In particular, the use of synthetic heteropolymers adsorbed onto the surface of a nanoparticle can yield selective detection of a molecular target. Synthetic molecular recognition has unique advantages in leveraging the photostability, versatility, and exceptional chemical stability of nanomaterials. In particular, single-walled carbon nanotubes (SWNT) exhibit a large Stokes shift and near infrared emission for maximum biological sample transparency. Optical biosensors with high signal transduction and molecular specificity can be synthesized with amphiphilic heteropolymers grafted to SWNT, and discovered by high-throughput screening. Herein, we describe the development and the characterization of surface-engineered nanoparticles, or synthetic antibodies, for protein detection.