MXene-Based Aptameric Fluorosensor for Sensitive and Rapid Detection of COVID-19 (Small 23/2023)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused COVID-19 pandemic has rapidly escalated into the largest global health emergency, which pushes to develop detection kits for the detection of COVID-19 with high sensitivity, specificity, and fast analysis. Here, aptamer-functionalized MXene nanosheet is demonstrated as a novel bionanosensor that detects COVID-19. Upon binding to the spike receptor binding domain of SARS-CoV-2, the aptamer probe is released from MXene surface restoring the quenched fluorescence. The performances of the fluorosensor are evaluated using antigen protein, cultured virus, and swab specimens from COVID-19 patients. It is evidenced that this sensor can detect SARS-CoV-2 spike protein at final concentration of 38.9 fg mL⁻¹ and SARS-CoV-2 pseudovirus (limit of detection: 7.2 copies) within 30 min. Its application for clinical samples analysis is also demonstrated successfully. This work offers an effective sensing platform for sensitive and rapid detection of COVID-19 with high specificity.     

Dielectrophoresis‐Based Protein Enrichment for a Highly Sensitive Immunoassay Using Ag/SiO2 Nanorod Arrays

A nanoscale insulator‐based dielectrophoresis (iDEP) technique is developed for rapid enrichment of proteins and highly sensitive immunoassays. Dense arrays of nanorods (NDs) by oblique angle deposition create a super high electric field gradient of 2.6 × 10²⁴ V² m^?3 and the concomitant strong dielectrophoresis force successfully traps small proteins at a bias as low as 5 V. 1800‐fold enrichment of bovine serum albumin protein at a remarkable rate of up to 180‐fold s^?1 is achieved using oxide coated Ag nanorod arrays with pre‐patterned sawtooth electrodes. Based on this system, an ultrasensitive immunoassay of mouse immunoglobulin G is demonstrated with a reduction in the limit of detection from 5.8 ng mL^?1 (37.6 pM) down to 275.3 fg mL^?1 (1.8 f M), compared with identical assays performed on glass plates. This methodology is also applied to detect a cancer biomarker prostate‐specific antigen spiked in human serum with a detection limit of 2.6 ng mL^?1. This high sensitivity results from rapid biomarker enrichment and metal enhanced fluorescence through the integration of nanostructures. The concentrated proteins also accelerate binding kinetics and enable signal saturation within 1 min. Given the easy fabrication process, this nanoscale iDEP system provides a highly sensitive detection platform for point‐of‐care diagnostics.     

Interfacial Assembly of Signal Amplified Multienzymes and Biorecognized Antibody into Proteinosome for an Ultrasensitive Immunoassay

Enzyme as signal tag has been widely employed in colorimetric immunoassays for decades. Nevertheless, it remains a great challenge to substantially improve the detection sensitivity of enzyme‐based immunoassays, which inhibits further critical applications. To circumvent this confinement, a multifunctional self‐assembled proteinosome based on the integration of signal amplification elements (enzyme) and biorecognition unit (antibody) is proposed for fabricating an immunoassay strategy with significantly enhanced sensitivity. Owing to the self‐assembly technique, this proteinosome not only efficiently loads abundant enzymes to possess high catalytic activity, but also enhances enzymatic stability and maintains recognition ability of antibody. Using imidacloprid as a model target, the proteinosome‐based immunoassay reaches a limit of detection down to the picogram mL^?1 level, which is 150‐fold lower than that of conventional enzyme‐linked immunosorbent assay. This method provides a versatile approach for constructing spherical proteinosome as a recognizer and amplifier for profiling a broad range of target antigen.     

Highly Fluorescent Magnetic Nanobeads with a Remarkable Stokes Shift as Labels for Enhanced Detection in Immunoassays

Fluorescence immunoassays are popular for achieving high sensitivity, but they display limitations in biological samples due to strong absorption of light, background fluorescence from matrix components, or light scattering by the biomacromolecules. A powerful strategy to overcome these problems is introduced here by using fluorescent magnetic nanobeads doped with two boron‐dipyrromethane dyes displaying intense emission in the visible and near‐infrared regions, respectively. Careful matching of the emission and absorption features of the dopants leads to a virtual Stokes shift larger than 150 nm achieved by an intraparticle Förster resonance energy transfer (FRET) process between the donor and the acceptor dyes. Additionally, the magnetic properties of the fluorescent beads allow preconcentration of the sample. To illustrate the usefulness of this approach to increase the sensitivity of fluorescence immunoassays, the novel nanoparticles are employed as labels for quantification of the widely used Tacrolimus (FK506) immunosuppressive drug. The FRET‐based competitive inhibition immunoassay yields a limit of detection (LOD) of 0.08 ng mL^?1, with a dynamic range (DR) of 0.15–2.0 ng mL^?1, compared to a LOD of 2.7 ng mL^?1 and a DR between 4.1 and 130 ng mL^?1 for the immunoassay carried out with direct excitation of the acceptor dye.     

Label‐Free Attomolar Detection of Proteins Using Integrated Nanoelectronic and Electrokinetic Devices

High‐sensitivity screening of biomarkers is critical to areas ranging from early disease detection and diagnosis to bioterrorism surveillance. Here the development of integrated nanoelectronic and electrokinetic devices for label‐free attomolar detection of proteins is reported. Electrically addressable silicon nanowire field‐effect transistors and electrodes for electrokinetic transport are integrated onto a common sensor chip platform, and the nanowire devices are subsequently functionalized with receptors for selective biomarker detection. Nanowire devices modified with monoclonal antibody for prostate specific antigen exhibit close to a 10⁴ increase in sensitivity due to streaming dielectrophoresis and corresponding electrostatic contribution to the binding affinity after application of an AC electric field. The devices are also modified with receptors for cholera toxin subunit B and achieve a similar enhancement. These results show general applicability of this method, and could open up opportunities in early stage disease detection and the analysis of proteins from single cells.     

Aminolated and Thiolated PEG‐Covered Gold Nanoparticles with High Stability and Antiaggregation for Lateral Flow Detection of Bisphenol A

The few lateral flow assays (LFAs) established for detecting the endocrine disrupting chemical bisphenol A (BPA) have employed citrate‐stabilized gold nanoparticles (GNPs), which have inevitable limitations and instability issues. To address these limitations, a more stable and more sensitive biosensor is developed by designing strategies for modifying the surfaces of GNPs with polyethylene glycol and then testing their effectiveness and sensitivity toward BPA in an LFA. Without the application of any enhancement strategy, this modified BPA LFA can achieve a naked‐eye limit of detection (LOD) of 0.8 ng mL^?1, which is 12.5 times better than the LOD of regular BPA LFAs, and a quantitative LOD of 0.472 ng mL^?1. This modified LFA has the potential to be applied to the detection of various antigens.     

High‐Throughput,Protein‐Targeted Biomolecular Detection Using Frequency‐Domain Faraday Rotation Spectroscopy

A clinically relevant magneto‐optical technique (fd‐FRS, frequency‐domain Faraday rotation spectroscopy) for characterizing proteins using antibody‐functionalized magnetic nanoparticles (MNPs) is demonstrated. This technique distinguishes between the Faraday rotation of the solvent, iron oxide core, and functionalization layers of polyethylene glycol polymers (spacer) and model antibody–antigen complexes (anti‐BSA/BSA, bovine serum albumin). A detection sensitivity of ≈10 pg mL^?1 and broad detection range of 10 pg mL^?1 ? c_BSA ? 100 µ g mL^?1 are observed. Combining this technique with predictive analyte binding models quantifies (within an order of magnitude) the number of active binding sites on functionalized MNPs. Comparative enzyme‐linked immunosorbent assay (ELISA) studies are conducted, reproducing the manufacturer advertised BSA ELISA detection limits from 1 ng mL^?1 ? c_BSA ? 500 ng mL^?1. In addition to the increased sensitivity, broader detection range, and similar specificity, fd‐FRS can be conducted in less than ≈30 min, compared to ≈4 h with ELISA. Thus, fd‐FRS is shown to be a sensitive optical technique with potential to become an efficient diagnostic in the chemical and biomolecular sciences.     

Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation

Microsphere-based immunoassay by flow cytometry has gained popularity lately in protein detection and infectious disease diagnosis due to its capacity for multiplexed analysis and simple assay format. Here, we demonstrated the power of microsphere-based immunoassay for high-sensitivity detection and accurate differentiation of influenza viruses. The effects of sample volume and bead number on the assay sensitivity of viral antigen detection were studied. Compared to enzyme-linked immunosorbent assays, flow-based bead assays provided approximately 10-fold lower detection limit for viral particle detection and performed similarly for recombinant viral hemagglutinin protein detection. A four-plexed assay for influenza virus typing and influenza B virus sublineage characterization was developed to demonstrate the potential for multiplexed viral antigen detection and differentiation.     

Sensitive Detection of Carcinoembryonic Antigen Using Stability‐Limited Few‐Layer Black Phosphorus as an Electron Donor and a Reservoir

The instability of few‐layer black phosphorus (FL‐BP) hampers its further applications. Here, it can be demonstrated that the instability of FL‐BP can also be the advantage for application in biosensor. First, gold nanoparticle/FL‐BP (BP‐Au) hybrid is facilely synthesized by mixing Au precursor with FL‐BP. BP‐Au shows outstanding catalytic activity (K = 1120 s^?1 g^?1) and low activation energy (17.53 kJ mol^?1) for reducing 4‐nitrophenol, which is attributed to the electron‐reservoir and electron‐donor properties of FL‐BP, and synergistic interaction of Au nanoparticles and FL‐BP. Oxidation of FL‐BP after catalytic reaction is further confirmed by transmission electron microscope, X‐ray photoelectron spectroscopy, and zeta potentials. Second, the catalytic activity of BP‐Au can be reversibly switched from “inactive” to “active” upon treatment with antibody and antigen in solution, thus providing a versatile platform for label‐free colorimetric detection of biomarkers. The sensor shows a wide detection range (1 pg mL^?1 to –10 µg mL^?1), high sensitivity (0.20 pg mL^?1), and selectivity for detecting carcinoembryonic antigen (CEA). Finally, the biosensor has been used to detect CEA in colon and breast cancer clinical samples with satisfactory results. Therefore, the instability of BP can also be the advantage for application in detecting cancer biomarker in clinic.     

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 10³ EID₅₀ mL^?1 (EID₅₀: 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme‐linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off‐chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low‐cost and convenient biomarker detection.     

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant

Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC₅₀ (inhibitory concentration) of free antigen was found to be 0.415 μg mL⁻¹ on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 μg mL⁻¹ with a limit of detection (LOD) of 0.05 μg mL⁻¹. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.     

Tracking Drug‐Induced Epithelial–Mesenchymal Transition in Breast Cancer by a Microfluidic Surface‐Enhanced Raman Spectroscopy Immunoassay

Epithelial–mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E‐cadherin and N‐cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed “SERS immunoassay”) based on sensitive and simultaneous detection of soluble E‐cadherin (sE‐cadherin) and soluble N‐cadherin (sN‐cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface‐enhanced Raman scattering readout to enable accurate detection of sE‐cadherin and sN‐cadherin from as low as 10 cells mL^?1. This assay enables tracking of a concurrent decrease in sE‐cadherin and increase in sN‐cadherin in breast cancer cells undergoing drug‐induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE‐cadherin and sN‐cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.     

Nanoprobe-based affinity mass spectrometry for selected protein profiling in human plasma

In recent decades, magnetic nanoparticles have emerged as a promising new platform in biomedical applications, particularly bioseparations. We have developed an immunoassay using antibody-conjugated magnetic nanoparticles as an efficient affinity probe to simultaneously preconcentrate and isolate targeted antigens from biological media. We combined this probe with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) to profile proteins in diluted human plasma. The nanoparticles were designed to detect several disease-associated proteins and could be used directly in MALDI MS without an elution step, thereby facilitating multiple antigen screening and the characterization of antigen variants. Plasma antigens bound rapidly (approximately 10 min) to the antibody-conjugated nanoparticles, allowing the assay to be performed within 20 min. With sensitivity of detection in the femtomole range, the nanoscale immunoassay is superior to assays using microscale particles. We applied our method to comparative protein profiling of patients with gastric cancer and healthy individuals and found differential protein expression levels associated with the disease as well as individuals. Given the flexibility of manipulating functional groups on the nanoprobes, their low cost, robustness, and simplicity of the assay, our approach shows promise for targeted proteome profiling in clinical settings.     

Amplification-Free Detection of SARS-CoV-2 Down to Single Virus Level by Portable Carbon Nanotube Biosensors (Small 34/2023)

The rapid and sensitive detection of trace-level viruses in a simple and reliable way is of great importance for epidemic prevention and control. Here, a multi-functionalized floating gate carbon nanotube field effect transistor (FG-CNT FET) based biosensor is reported for the single virus level detection of SARS-CoV-2 virus antigen and RNA rapidly with a portable sensing platform. The aptamers functionalized sensors can detect SARS-CoV-2 antigens from unprocessed nasopharyngeal swab samples within 1 min. Meanwhile, enhanced by a multi-probe strategy, the FG-CNT FET-based biosensor can detect the long chain RNA directly without amplification down to single virus level within 1 min. The device, constructed with packaged sensor chips and a portable sensing terminal, can distinguish 10 COVID-19 patients from 10 healthy individuals in clinical tests both by the RNAs and antigens by a combination detection strategy with an combined overall percent agreement (OPA) close to 100%. The results provide a general and simple method to enhance the sensitivity of FET-based biochemical sensors for the detection of nucleic acid molecules and demonstrate that the CNT FG FET biosensor is a versatile and reliable integrated platform for ultrasensitive multibiomarker detection without amplification and has great potential for point-of-care (POC) clinical tests.     

Photonic-Plasmonic Coupling Enhanced Fluorescence Enabling Digital-Resolution Ultrasensitive Protein Detection

Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal-to-noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52-fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL⁻¹ and 100 fg mL⁻¹ in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays.     

Multiplexed immunoassay: quantitation and profiling of serum biomarkers using magnetic nanoprobes and MALDI-TOF MS

Taking advantage of efficient affinity extraction by surface-functionalized magnetic nanoparticles (MNPs) and accurate MALDI-TOF MS readout, we present a multiplexed immunoassay for simultaneous enrichment and quantitation of multiple disease-associated antigens, serum amyloid A (SAA), C-reactive protein (CRP), and serum amyloid P (SAP) from human serum. To obtain reproducible MALDI signal response with direct on-MNP detection, the seed-layer method improved homogeneity of the cocrystallization of MNPs and captured antigens. Our methodology demonstrated good quantitation linearity of targeted analytes (R(2) approximately 0.97) with reduced signal variation (RSD < 10%). The lower limit of quantitation is in the nanogram level with overall assay precision (intraday, 7.0%; interday, 11.3%) and accuracy (intraday, 6.3%; interday, 17.5%) including steps of nanoprobe extraction and MALDI-TOF MS analysis. This triplexed immunoassay showed overexpression of SAA and CRP in patients with cardiac catheterization or gastric cancer (P < 0.05), consistent with single-analyte ELISA and previous studies. Compared to the determination of disease onset by single protein quantitation, our multiplexed immunoassay revealed a distinct triplexed pattern in the control group, patients with gastric cancer, and cardiac catheterization. On the basis of the advantages of flexibility in nanoprobe preparation, high specificity and sensitivity, and rapid screening by MALDI-TOF MS, this platform may provide a new methodology for disease diagnosis.     

Au@gap@AuAg Nanorod Side‐by‐Side Assemblies for Ultrasensitive SERS Detection of Mercury and its Transformation

As one of the most toxic heavy metal elements, mercury ion (Hg²⁺) and its methylated product, methylmercury (MeHg) can pose a threat to human health and the environment. Herein, a novel Raman biosensor with cascade sensitivity is developed for Hg²⁺ detection through Au@gap@AuAg nanorod side‐by‐side assemblies. Due to the strong electromagnetic coupling from the assemblies and core–shell structure, the Raman sensor possesses high sensitivity with the limit of detection (LOD) of 0.001 ng mL^‐1, which is about one order lower than traditional atomic fluorescence spectrometer (AFS) methods. Moreover, the fabricated biosensor is used to measure residual mercury levels in tissues and eggs of hens fed high‐mercury diets, and the results show total mercury in collected egg yolks is 20 times higher than whites. Furthermore, the form of mercury in the eggs is also analyzed by high‐performance liquid chromatography coupled with AFS, and, unexpectedly, the methylated product MeHg tends to only be found in egg whites. These interesting differences may indicate a new research direction for the toxicity of mercury in living organisms, and the developed ultrasensitive Surface Enhanced Raman Scattering (SERS) method could pave a broad way for the application of biosensors in Hg detection.     

A Rapid and Robust Diagnostic for Liver Fibrosis Using a Multichannel Polymer Sensor Array

Liver disease is the fifth most common cause of premature death in the Western world, with the irreversible damage caused by fibrosis, and ultimately cirrhosis, a primary driver of mortality. Early detection of fibrosis would facilitate treatment of the underlying liver disease to limit progression. Unfortunately, most cases of liver disease are diagnosed late, with current strategies reliant on invasive biopsy or fragile lab‐based antibody technologies. A robust, fully synthetic fluorescent‐polymer sensor array is reported, which, rapidly (in 45 minutes), detects liver fibrosis from low‐volume serum samples with clinically relevant specificity and accuracy, using an easily readable diagnostic output. The simplicity, rapidity, and robustness of this method make it a promising platform for point‐of‐care diagnostics for detecting and monitoring liver disease.     

Carbon Nanotubes Multifunctionalized by Rolling Circle Amplification and Their Application for Highly Sensitive Detection of Cancer Markers

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)‐based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single‐strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano‐biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha‐fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme‐linked immunosorbent assay (ELISA), and its concentration range is 10 000 times wider. Control experiments show that as low as 10 pg mL^?1 AFP or PSA could be detected even in the presence of interfering protein markers with a more than 10⁵‐fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL^?1 (70 aM) and 10 fg mL^?1 (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.     

Electrochemical detection of prostate-specific antigen based on gold colloids/alumina derived sol-gel film

An electrochemical immunosensor for total prostate-specific antigen (PSA) was fabricated based on anti-PSA antibody-functionalized gold colloids/alumina sol-gel film. The presence of colloidal gold provides a good microenvironment for the immobilization of biomolecules. The fabrication process of the immunosensor was characterized by cyclic voltammetry. A direct electrochemical immunoassay format was employed to detect PSA on the basis of the potential change before and after the antigen-antibody interaction. Under optimal conditions, the potential change was proportional to PSA concentration ranging from 4.0 to 13 ng mL^− 1 with a detection limit of 3.4 ng mL^− 1. In addition, the performance and factors influencing the performance of the immunosensor were investigated and optimized.