Complex Serum Samples Research Articles

Target-promoted catalytic DNA molecular circuit network coupled with endonuclease amplification for sensitive and label-free electrochemical aptamer antibiotic assay

Sarafloxacin (SAR) is a common antibiotic that accumulates in animal tissues, which potentially poses health risks to humans via ingestion. Sensitive detection of SAR in animal-derived foods is therefore critical. Traditional SAR detection methods based on HPLC or antibody immunoassays encounter the limitations of cost, sensitivity and operational simplicity. In this study, with the design of a new DNAzyme/catalytic hairpin assembly (CHA) molecular circuit network coupled with endonuclease amplification, we developed an electrochemical aptamer (AP) biosensor with high sensitivity for label-free SAR detection in milk. The sensor functions by specific binding of SAR to APs, which release DNAzyme sequences to initiate the catalytic DNAzyme/CHA molecular circuit network amplification for forming many ssDNAs and dsDNA duplexes. These sequences further hybridize with hairpin signal probes on sensor electrode to create favorable nicking sites for endonuclease, which cleaves hairpin signal probes to liberate lots of G-quadruplex strands to complex with and capture hemin. Subsequent hemin reduction by electrochemistry thus results in dramatically enhanced currents to achieve label-free detection of SAR in linear range between 10 pM and 80 nM with detection limit of 1.62 pM. Our sensor also shows satisfactory reproducibility, repeatability and stability, demonstrates high selectivity for the target SAR against other interfering antibiotics, e.g., norfloxacin, penicillin and gentamicin and can realize detection of low concentrations of SAR in diluted complex milk and human serum samples, thereby underscoring its promises for convenient and sensitive assay of different trace antibiotics.

Multichannel sensor array based on carbon dots and metal ions system for physiological phosphates sensing

The detection of physiological phosphates is crucial for elucidating metabolic processes and related diagnoses of diseases. Herein, a carbon dots (CDs) modulated multichannel fluorescent sensor array was proposed for detecting and distinguishing physiological phosphates. In this sensor, three metal ions (Nd3+, Ag+, and Fe3+) with significantly different binding forces with phosphate ions were selected to trigger fluorescence quenching of three CDs (BCDs, GCDs, and RCDs). Upon adding physiological phosphates, the fluorescence intensity of CDs changed. Depending on the discrepancy in the content of phosphate units in different physiological phosphates, the sensor array realized the detection and differentiation of five physiological phosphates related to adenosine triphosphate (ATP) hydrolysis, and has good anti-interference ability, which can realize the differentiation of physiological phosphates in complex serum samples.

Fabrication of a highly sensitive conductive copolymer layer modified immunosensing tool for cytokeratin-19 fragment detection

Design of a new and highly selective impedimetric biosensor based on the immobilization of a fragment of cytokeratin subunit 19 (CYFRA 21-1)-specific antibodies onto a conductive copolymer layer (poly(epoxy-substituted thiophene)-co-poly(3,4-ethylenedioxythiophene), CCL)-coated indium tin oxide (ITO) for the determination of CYFRA 21-1 in serum samples was reported. CCL was applied as a working electrode-modifying reagent to prepare the surface of the working ITO electrode, and CYFRA 21-1-specific antibodies bound to epoxy ends present in the copolymer layer matrix. The impedimetric response of the immunosensor to CYFRA 21-1 was recorded, and the signaling mechanism of the suggested immunosensor was based on an increase in the charge transfer resistance in the presence of target CYFRA 21-1. The developed biosensor exhibited excellent selectivity for CYFRA 21-1, and it could detect CYFRA 21-1 in a linear dynamic range from 0.0125 to 35 pg/mL with a limit of quantification (LOQ) of 12.25 fg/mL. The designed biosensor showed good storage stability, high sensitivity, suitable repeatability and reproducibility, and acceptable long-term stability. The clinical analysis of the manufactured immunosensor was explored for the analysis of serum samples, and high recoveries and good relative standard deviations (RSDs) were obtained in the range of 97.01 and 103.17 %, respectively. This copolymer-based biosensor could be a feasible tool for clinical diagnosis in complex serum samples.

Parallel optofluidic detection of multiple cardiac biomarkers for point-of-care testing applications

Point-of-care testing (POCT), which enables personalized, accurate, affordable, and accessible medical services, is indispensable in the diagnostics, screening, and treatment of disease, especially for emergency and severe cases. Considering the complexity of disease, multiplexed point-of-care (MPOC) testing is very important for the simultaneous detection of several biomarkers to improve accuracy and increase analysis throughput. In this work, a microfiber interferometer based multiplexed optofluidic (MIMO) chip sensor is proposed for multiplexed, quantitative detection of cardiac biomarkers. Four microfiber interferometers are suspended and fixed in four separate microscale sample channels of an imprinted polydimethylsiloxane (PDMS) chip. A drop of the sample is distributed into the four parallel channels automatically and reacts with the probe on the microfiber sensor surface specifically, which leads to the wavelength shift of the spectrum resulting from evanescent wave interactions with biomolecules. There microfiber interferometers are functionalized with myoglobin (Myo) antibody, cardiac troponin I (cTnI) antibody, and creatine kinase–myocardial band isoenzymes (CK–MB) antibody respectively and the rest microfiber interferometer is with no functionalization, which acts as the control channel to reduce noise. By using compact dedicated commercial optical modules, miniaturized and low cost experimental setup is organized and achieve four channel simultaneous measurement through time division multiplexing. Three cardiac biomarkers, Myo, cTnI, and CK–MB, are synchronously detected on the proposed MIMO chip sensor. In a standard buffer sample, the log-linear sensing range of CK–MB is 0.2 ∼ 200 ng/mL with detection limit of 3.7 pg/mL, the log-linear sensing range of Myo is 2 ∼ 2000 ng/mL with detection limit of 0.5 ng/mL, and the log-linear sensing range of cTnI 0.02 ∼ 20 ng/mL with detection limit of 2.6 pg/mL, which all perfectly cover the actual diagnostic limit of myocardial infarction. Furthermore, the MIMO chip sensor maintains excellent performance of both specificity and detection capabilities in more complex serum samples. In double-blind trials of multiple biomarker detection, the MIMO chip sensor exhibits comparable detection ability with a commercial kit and good resistance to environmental disturbance. The proposed MIMO chip sensor sensing system offers a low-cost, portable, self-driven, and miniaturized scheme for a multiplexed point-of-care testing device, promoting the feasibility and accessibility of personalized medical services and paving the way for clinical application and industrialization.

A Versatile One-Step Enzymatic Strategy for Efficient Imaging and Mapping of Tumor-Associated Tn Antigen.

Tn antigen (CD175), recognized as the precursor monosaccharide (α-GalNAc) of mucin O-glycan, is a well-known tumor-associated carbohydrate antigen (TACA). It has emerged as a potential biomarker for cancer diagnosis and prognosis. However, the role it plays in cancer biology remains elusive due to the absence of a sensitive and selective detection method. In this study, we synthesized two new probes based on a unique uridine-5'-diphospho-α-d-galactose (UDP-Gal) derivative, each functionalized with either a fluorescence or a cleavable biotin tag, to develop an innovative one-step enzymatic labeling strategy, enabling the visualization, enrichment, and site-specific mapping of the Tn antigen with unparalleled sensitivity and specificity. Our versatile strategy has been successfully applied to detect and image Tn antigen across various samples, including the complex cell lysates, live cells, serum, and tissue samples. Compared to the traditional lectin method, this one-step enzymatic method is simpler and more efficient (>10/100-fold in sensitivity). Furthermore, it allowed us to map 454 Tn-glycoproteins and 624 Tn-glycosylation sites from HEK293FTn+ and Jurkat cells. Therefore, our strategy provides an exceptionally promising tool for revealing the biological functions of the Tn antigen and advancing cancer diagnostics.

Rapid redox-response featured visual ascorbic acid sensor based on simple-assembled europium metal-organic framework

A facile, fast and visible sensing platform for ascorbic acid (AA) detection has been developed based on self-assembled hydrangea-like europium metal-organic framework (HL-EuMOF). HL-EuMOF was synthesized through a simple one-step mixing process with Eu3+ and 1, 10-phenanthroline-2, 9-dicarboxylic acid at room temperature, which exhibited excellent properties including strong red fluorescence, long decay lifetime (548.623 μs) and good luminescent stability. Based on the specific redox reaction between Fe3+ and AA, the HL-EuMOF@Fe3+ was fabricated with “turn-off” response for AA, where the resulting Fe2+ displayed effective fluorescence quenching ability toward HL-EuMOF. The sensor demonstrated low detection limit (31.94 nM), rapid response time (30 s) and high selectivity. Integration of smartphone-assisted RGB analysis with HL-EuMOF@Fe3+ permitted convenient and visible quantitative determination of AA level. This approach also presented good detection performances in complex human serum and beverage samples, which could provide a valuable tool for AA detection in biomedical research and food industry.

PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

APyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied toALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapidanalysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

Magnetic beads and GO-assisted enzyme-free signal amplification fluorescent biosensors for disease diagnosis

Cancer detection is still a major challenge in public health. Identification of oncogene is the first step toward solving this problem. Studies have revealed that various cancers are associated with miRNA expression. Therefore, the sensitive detection of miRNA is substantially important to solve the cancer problem. In this study, let-7a, a representative substance of miRNA, was selected as the detection target. With the assistance of magnetic beads commonly used in biosensors and self-synthesized graphene oxide materials, specificity and sensitivity detection of the target gene let-7a were achieved via protease-free signal amplification. The limit of detection (LOD) was as low as 15.015pM. The fluorescence signal intensity showed a good linear relationship with the logarithm of let-7a concentration. The biosensor could also detect let-7a in complex human serum samples. Overall, this fluorescent biosensor is not only simple to operate, but also strongly specificity to detect let-7a. Therefore, it has substantial potential for application in the early diagnosis of clinical medicine and biological research.

An electrochemical biosensor for the amplification of thrombin activity by perylene-mediated photoinitiated polymerization

BackgroundThrombin, a coagulation system protease, is a key enzyme involved in the coagulation cascade and has been developed as a marker for coagulation disorders. However, the methods developed in recent years have the disadvantages of complex operation, long reaction time, low specificity and sensitivity. Meanwhile, thrombin is at a lower level in the pre-disease period. Therefore, to accurately diagnose the disease, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification technology. ResultsWe designed an electrochemical biosensor based on photocatalytic atom transfer radical polymerization (photo-ATRP) signal amplification for the detection of thrombin. Sulfhydryl substrate peptides (without carboxyl groups) are self-assembled to the gold electrode surface via Au–S bond and serve as thrombin recognition probes. The substrate peptide is cleaved in the presence of thrombin to generate –COOH, which can form a carboxylate-Zr(IV)-carboxylate complex via Zr(IV) and initiator (α-bromophenylacetic acid, BPAA). Subsequently, an electrochemical biosensor was prepared by introducing polymer chains with electrochemical signaling molecules (ferrocene, Fc) onto the electrode surface by photocatalytic (perylene, Py) mediated ATRP using ferrocenylmethyl methacrylate (FMMA) as a monomer. The concentration of thrombin was evaluated by the voltammetric signal generated by square wave voltammetry (SWV), and the result showed that the biosensor was linear between 1.0 ng/mL ∼ 10 fg/mL, with a lower detection limit of 4.0 fg/mL (∼0.1 fM). Moreover, it was shown to be highly selective for thrombin activity in complex serum samples and for thrombin inhibition screening. SignificanceThe biosensor is an environmentally friendly and economically efficient strategy while maintaining the advantages of high sensitivity, anti-interference, good stability and simplicity of operation, which has great potential for application in the analysis of complex samples.

Carbon dot-based fluorescent probe for early diagnosis of pheochromocytoma through identification of circulating tumor cells

Pheochromocytoma (PCC), as a rare neuroendocrine tumor, is often missed or misdiagnosed because of its atypical clinical manifestations. To realize the early accurate diagnosis of PCC, we have selected circulating tumor cells (CTCs) with more complete biological information as biomarkers and developed a simple and novel fluorescence cytosensor. Octreotide-2,2′,2′′,2′’’- (1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) modified magnetic Fe3O4 and signal amplification CDs@SiO2 nanospheres are prepared to capture and detect PCC-CTCs from peripheral blood via binding to the somatostatin receptor SSTR2 overexpressed on the surface of PCC cells. During the detection process, the target cells were separated and enriched by magnetic capture probes (Fe3O4-DOTA), and then signal probes (CDs@SiO2-DOTA) could also specifically bound to target cells to form the sandwich-like structure for fluorescence signal output. The proposed fluorescence cytosensor has revealed good sensitivity and selectivity for quantitative analysis of PCC-CTCs in the concentration of 5–1000 cells mL−1 with a LOD of 2 cells mL−1. More importantly, designed fluorescence cytosensor has shown good reliability and stability in complex serum samples. This strategy provides a new way for detection of PCC-CTCs.

An ultrasensitive biosensor with suppressed background signals for FEN1 detection in a homogeneous reaction via cascade primer exchange reaction and CRISPR/Cas12a system

Flap endonuclease 1 (FEN1) is a structure-specific enzyme which plays a crucial role in DNA replication and repair within mammalian cells. FEN1 is also known to be overexpressed in various cancer cells and has emerged as a potential biomarker for cancer diagnosis. However, there have been limited progress in development of sensitively analytical methods toward FEN1 detection. Herein, we have developed an ultrasensitive FEN1 sensing biosensor for FEN1 detection by combining primer exchange reaction (PER) with CRISPR/Cas12a signal amplification (PCSA). This innovative method achieves remarkable sensitivity for FEN1 detection, with a limit of detection (LOD) as low as 3.72 × 10−7 U/μL. Notably, PCSA effectively suppresses background signals, resulting in a high signal-to-noise ratio exceeding 31-fold due to three specific enzymatic recognition reactions. Consequently, PCSA demonstrates outstanding selectivity and robust anti-interference capabilities. The PCSA was employed to detect the FEN1 in complex serum samples, as well as in normal and tumor cells, achieving an LOD for FEN1 as low as in 10 cancer cells. Additionally, PCSA offers a simple operational process and yields a visually observable signal output, simplifying its use for the point-of-care testing (POCT) in some resource-limited regions. This straightforward approach holds significant promise for broad applications in FEN1 sensing and inhibitor screening.

Proximity Amplification-Enabled Electrochemical Analysis of Tumor-Associated Glycoprotein Biomarkers.

Glycoproteins produced and secreted from specific cells and tissues are associated with several diseases and emerge as typical biomarkers to provide useful information in cancer diagnosis considering their abnormal expression levels. In this work, we design a universal method to achieve the accurate and sensitive analysis of tumor-associated glycoprotein biomarkers based on both carbohydrate recognition and protein recognition at the same protein surface. The byproduct of dual recognition-induced proximity amplification, pyrophosphate, triggers the disassembly of methylene blue-encapsulated metal-organic frameworks, MB@ZIF-90. As a result, methylene blue molecules are released to arouse amplified electrochemical responses for glycoprotein analysis. Experimental results demonstrate the high-accuracy analysis of carcinoembryonic antigen, a typical glycoprotein biomarker in cancer diagnosis, in a linear range of 0.001-100 ng mL-1 with a low limit of detection of 0.419 pg mL-1. The method also displays satisfactory specificity and recoveries in complex serum samples and proves good versatility by adopting two other tumor-associated glycoprotein biomarkers, α-fetoprotein and mucin-1, as the targets. Therefore, this work provides a valuable tool for the analysis of glycoprotein biomarkers, which may be of great potential in early warning of malignant tumors in clinical applications.

CRISPR/Cas12a and primer-assisted rolling circle amplification integrated ultra-sensitive dual-signal sensing platform for EGFR 19 detection

Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.

Engineered peptide-cell membrane interfaces for ultrasensitive and selective detection of ERBB2

Biosensors hold great promise for rapid clinical diagnosis of cancer at a curable stage of the disease. However, traditional bioassays face challenges such as instability and low selectivity when applied to complex samples. Herein, inspired by the structure and functions of natural cell membranes, we fabricated an ultrasensitive and selective sensor of human epidermal growth factor receptor 2 (ERBB2) in serum based on the engineered peptide membrane interfaces. The polyethylene glycol (PEG) and its derivatives fixed on the Au surface as support layers, on which the phospholipid and multifunctional peptides are applied to construct a hydration layer for antifouling. The engineered peptide membrane interfaces forming a structure with flow recognition, high hydrophilicity and stability. The low nonspecific adsorption at the cell membrane interface based on engineered peptides was achieved by PEG and its derivatives as well as engineered peptide membrane, enabling selective detection in complex serum samples. The fabricated sensor demonstrates good stability and a remarkable limit of detection value of 0.21 ng/mL for ERBB2. Thus, there is great potential for the application of this engineered peptide-cell membrane strategy in further biomimetic interface construction in cell or protein analysis.

Dual-model photoelectrochemical biosensor via DNAzyme walker integrated nanoprobe for ultrasensitive ratiometric detection of microRNA-155

Ultrasensitive detection for low concentration and abnormal expression of microRNA-155 (miR-155) in serum (fM level) at the early stage of cancer still exist great challenges. Therefore, a dual-model photoelectrochemical biosensor was developed based on label-free trimetallic nanoprobe and Mg2+ dependent DNAzyme driven DNA walker to generate ratiometric dual-signal output for sensitive biosensing of miR-155 from breast cancer cells (MCF-7) and cervical cancer cells (HeLa). In signal amplification strategy, PtCo@Prussian Blue nanozyme was selected as a signal nanoprobe with high catalytic activity and large specific surface area, while two dimensional MXene/AuNPs as surface modification material was applied to improve the active sites of the electrode. With the presence of the target, the DNAzyme assisted DNA walker process was initiated and abundant signal nanoprobe was introduced to the electrode surface, producing high current response and weak ultraviolet-visible spectrophotometry (UV–vis) response. Hence, the biosensor for miR-155 shows a low limit of detection of 1.2 fM from the linear range of 5 fM to 5 nM, exhibiting excellent stability and reproducibility. Moreover, the proposed biosensor demonstrates superior performance for miR-155 in complex human serum samples and tumor cell lysates, presenting great application potential for early-stage clinical diagnosis.

A cloth-based single-working-electrode electrochemiluminescence sensor for simultaneous detection of diabetes complication markers.

As one of the most common noninfectious diseases, diabetes and diabetic complications (DDC) have attracted great attention in the field of life and health. However, simultaneous detection of DDC markers usually requires labor- and time-consuming steps. Here, a novel cloth-based single-working-electrode electrochemiluminescence (SWE-ECL) sensor was designed for the simultaneous detection of multiple DDC markers. For this sensor, three independent ECL cells are distributed on the SWE, which is a simplification of the configuration of traditional sensors for simultaneous detection. In this way, the modification processes and ECL reactions occur at the back of the SWE, eliminating the adverse effects caused by human intervention on the electrode. Under optimized conditions, glucose, uric acid and lactate were determined, with corresponding linear dynamic ranges of 80-4000μM, 45-1200μM and 60-2000μM, and detection limits of 54.79μM, 23.95μM and 25.82μM, respectively. In addition, the cloth-based SWE-ECL sensor exhibited good specificity and satisfactory reproducibility, and its actual application potential was verified by measuring complex human serum samples. Overall, this work developed a simple, sensitive, low-cost and rapid method for the simultaneous quantitative determination of multiple markers related to DDC and demonstrated a new route for multiple-marker detection.

Biocompatible epoxysilane substituted polymer-based nano biosensing platform for label-free detection of cancer biomarker SP17 in patient serum samples

Herein, we report the results of the studies relating to developing a simple, sensitive, cost-effective, and disposable electrochemical-based label-free immunosensor for real-time detection of a new cancer biomarker, sperm protein-17 (SP17), in complex serum samples. An indium tin oxide (ITO) coated glass substrate modified with self-assembled monolayers (SAMs) of 3-glycidoxypropyltrimethoxysilane (GPTMS) was functionalized via covalent immobilization of monoclonal anti-SP17 antibodies using EDC(1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride) - NHS (N-hydroxy succinimide) chemistry. The developed immunosensor platform (BSA/anti-SP17/GPTMS@SAMs/ITO) was characterized via scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA), Fourier transform infrared (FT-IR) spectroscopic, and electrochemical techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques. The fabricated BSA/anti-SP17/GPTMS@SAMs/ITO immunoelectrode platform was used to measure changes in the magnitude of the current of the electrodes through an electrochemical CV and DPV technique. A calibration curve between current and SP17 concentrations exhibited a broad linear detection range of (100–6000 & 50–5500 pg mL−1), with enhanced sensitivity (0.047 & 0.024 μA pg mL−1 cm−2), limit of detection (LOD) and limit of quantification (LOQ) of 47.57 & 142.9 pg mL−1 and 158.58 & 476.3 pg mL−1, by CV and DPV technique, respectively with a rapid response time of 15 min. It possessed exceptional repeatability, outstanding reproducibility, five-time reusability, and high stability. The biosensor's performance was evaluated in human serum samples, giving satisfactory findings obtained via the commercially available enzyme-linked immunosorbent assay (ELISA) technique, proving the clinical applicability for early diagnosis of cancer patients. Moreover, various in vitro studies in murine fibroblast cell line L929 have been performed to assess the cytotoxicity of GPTMS. The results demonstrated that GPTMS has excellent biocompatibility and can be used for biosensor fabrication.

Ultrasensitive Detection of Multidrug-Resistant Bacteria Based on Boric Acid-Functionalized Fluorescent MOF@COF.

The widespread use of antibiotics has made multidrug-resistant bacteria (MDRB) one of the greatest threats toward global health. Current conventional microbial detection methods are usually time-consuming, labor-intensive, expensive, and unable to detect low concentrations of bacteria, which cause great difficulties in clinical diagnosis and treatment. Herein, we constructed a versatile biosensing platform on the basis of boric acid-functionalized porous framework composites (MOF@COF-BA), which were able to realize highly efficient and sensitive label-free MDRB detection via fluorescence. In this design, MDRB were captured using aptamer-coated nanoparticles and the fluorescent probe MOF@COF-BA was tightly anchored onto the surface of MDRB due to interactions between boric acid groups and glycolipids on bacteria cells. Benefitting from the remarkable fluorescence performance of MOF@COF-BA, rapid and specific detection of MDRB, such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB), was realized with a detection range of 20-108 CFU/mL (for both) and limits of detection of 7 CFU/mL (MRSA) and 5 CFU/mL (AB). The feasibility of using the developed platform to selectively detect MRSA and AB from complex urine, human serum, and cerebrospinal fluid samples was also demonstrated. This work provides a promising strategy for accurate MDRB diagnosis, avoiding serious infection using rational antibiotic therapy.

Ratiometric electrochemical OR gate assay for NSCLC-derived exosomes

Non-small cell lung cancer (NSCLC) is the most common pathological type of LC and ranks as the leading cause of cancer deaths. Circulating exosomes have emerged as a valuable biomarker for the diagnosis of NSCLC, while the performance of current electrochemical assays for exosome detection is constrained by unsatisfactory sensitivity and specificity. Here we integrated a ratiometric biosensor with an OR logic gate to form an assay for surface protein profiling of exosomes from clinical serum samples. By using the specific aptamers for recognition of clinically validated biomarkers (EpCAM and CEA), the assay enabled ultrasensitive detection of trace levels of NSCLC-derived exosomes in complex serum samples (15.1 particles μL−1 within a linear range of 102–108 particles μL−1). The assay outperformed the analysis of six serum biomarkers for the accurate diagnosis, staging, and prognosis of NSCLC, displaying a diagnostic sensitivity of 93.3% even at an early stage (Stage I). The assay provides an advanced tool for exosome quantification and facilitates exosome-based liquid biopsies for cancer management in clinics.Graphical

Microporous PdCuB nanotag-based electrochemical aptasensor with Au@CuCl2 nanowires interface for ultrasensitive detection of PD-L1-positive exosomes in the serum of lung cancer patients

Programmed cell death ligand 1 protein-positive (PD-L1+) exosomes have been found to be a potential biomarker for the diagnosis of non-small cell lung cancer (NSCLC). However, the development of highly sensitive detection technique for PD-L1+ exosomes is still a challenge in clinical applications. Herein, a sandwich electrochemical aptasensor based on ternary metal-metalloid palladium-copper-boron alloy microporous nanospheres (PdCuB MNs) and Au@CuCl2 nanowires (NWs) was designed for the detection of PD-L1+ exosomes. The excellent peroxidase-like catalytic activity of PdCuB MNs and the high conductivity of Au@CuCl2 NWs endow the fabricated aptasensor with intense electrochemical signal, thus enabling the detection of low abundance exosomes. The analytical results revealed that the aptasensor maintained favorable linearity over a wide concentration range of 6 orders of magnitude and reached a low detection limit of 36 particles/mL. The aptasensor is successfully applied to the analysis of complex serum samples and achieves the accurate identification of clinical NSCLC patients. Overall, the developed electrochemical aptasensor provides a powerful tool for early diagnosis of NSCLC.