Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

Quantum dot-based fluorescence immunosorbent assay for the rapid detection of bacitracin zinc in feed samples.

Bacitracin zinc (BAC), a polypeptide antibiotic, is utilized as a feed additive due to its ability to promote growth in animals. However, the abuse of BAC can lead to a great threat to food safety. Therefore, there is an urgent need to develop a rapid and sensitive detection method. In this study, a monoclonal antibody (mAb) against BAC with excellent sensitivity and specificity was obtained. For the first time, quantum dots (QDs) were conjugated with the prepared mAb against BAC and rabbit anti-mouse antibody to fabricate a direct and an indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) to detect BAC. The IC50 of dc-FLISA and ic-FLISA were 0.28 ng/ml and 0.17 ng/ml, respectively. The limits of detection were 0.0016 ng/ml and 0.001 ng/ml, respectively, and the detection ranges were 0.0016-46.50 ng/ml and 0.001-35.65 ng/ml, respectively. In addition, the recovery rate of the two methods ranged from 93.5% to 112.0%, and the coefficient of variation (CV) was less than 10%. Therefore, the methods developed in this work have the merits of low cost, simple operation, and high sensitivity, which provide an effective analytical tool for BAC residue detection in feed samples.

Mechanically transduced immunosorbent assay to measure protein-protein interactions.

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.

Nanobody-loaded immunosorbent for highly-specific removal of interleukin-17A from blood.

Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.55 mg/g gel, which is almost consistent with the saturated adsorption capacity determined in dynamic adsorption. The in vitro plasma perfusion test demonstrated a remarkable adsorptive performance of the Nb-coupled sorbent since more than 75% IL-17A could be eliminated under the plasma/sorbent ratio of 1000:1. These results indicated the Nb-loaded immunosorbent can provide a simple and economic platform technology for immunoaffinity depletion of single or even multiple cytokines from plasma.

Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1-105.3% and coefficients of variation of 4.7-9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples' data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

An In Situ Generated Prussian Blue Nanoparticle-Mediated Multimode Nanozyme-Linked Immunosorbent Assay for the Detection of Aflatoxin B1.

This work aims to develop a novel multimode (photothermal/colorimetric/fluorescent) nanozyme-linked immunosorbent assay (NLISA) based on the in situ generation of Prussian blue nanoparticles (PBNPs) on the surface of magnetic nanoparticles (MNPs). Being considered the most toxic among the mycotoxins, aflatoxin B1 (AFB1) was chosen as the proof-of-concept target. In this strategy, MNPs, on which an AFB1 aptamer was previously assembled via streptavidin-biotin linkage, are anchored to 96-well plates by AFB1 and antibody. In the presence of HCl and K4Fe(CN)6, PBNPs formed in situ on the MNP surface, thereby achieving photothermal and colorimetric signal readout due to their photothermal effect and intrinsic peroxidase-like activity. Based on fluorescence quenching by MNPs, Cy5 fluorescence was recovered by the in situ generation of PBNPs to facilitate ultrasensitive fluorescence detection. Photothermal and colorimetric signals allow portable/visual point-of-care testing, and fluorescent signals enable accurate determination with a detection limit of 0.54 fg/mL, which is 6333 and 28 times lower than those of photothermal and colorimetric analyses, respectively. We expect that this proposed multimode NLISA can not only reduce the false-positive/negative rates through the multisignal crossdetection in AFB1 monitoring but also provide a universal way of sophisticated instrumentation-free, easy-to-use, cost-effective, and highly sensitive detection of other food hazards.

Peptide Linker Affecting the Activity Retention Rate of VHH in Immunosorbents.

VHH-based immunosorbents are an emerging and promising tool for the removal of toxic substances from plasma. However, the small size of VHHs is a double-edged sword, bringing both benefits and drawbacks to the immunosorbent. The small size of the VHH allows a higher coupling density, while the closer distance to the resin might create steric hindrance for paratope access. The latter could be avoided by inserting a linker between the VHH and the gel attachment site. Here, we report an approach to improve the activity retention of the immobilized VHH by selecting suitable linkers between the VHH and the site-specific immobilization site on the resin. Seven peptide linkers differing in length and flexibility were fused to the VHH and contained the formylglycine generating enzyme (FGE) recognition sequence. These constructs were expressed in the cytoplasm of bacteria and purified, the VHH production yield and affinity for its cognate antigen was measured. Furthermore, the fGly conversion, the immobilization of the aldehyde-containing nanobodies, the immobilization on resin and the antigen binding activity of the VHH-based immunoadsorbents was monitored. The VHH with longer and rigid, proline-rich linkers exhibited good expression yield of approximately 160 mg/L of culture, a fGly conversion of up to 100%, and the highest activity retention rate of more than 68%. This study unveiled two suitable linkers for the preparation of VHH-based immunosorbents that will assist the development of their clinical application.

A rapid and highly sensitive biomarker detection platform based on a temperature-responsive liposome-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) is widely used in various fields to detect specific biomarkers. However, ELISA tests have limited detection sensitivity (≥ 1 pM), which is insufficiently sensitive for the detection of small amounts of biomarkers in the early stages of disease or infection. Herein, a method for the rapid and highly sensitive detection of specific antigens, using temperature-responsive liposomes (TLip) containing a squaraine dye that exhibits fluorescence at the phase transition temperature of the liposomes, was developed. A proof-of-concept study using biotinylated TLip and a streptavidin-immobilized microwell plate showed that the TLip bound to the plate via specific molecular recognition could be distinguished from unbound TLip within 1 min because of the difference in the heating time required for the fluorescence emission of TLip. This system could be used to detect prostate specific antigen (PSA) based on a sandwich immunosorbent assay using detection and capture antibodies, in which the limit of detection was as low as 27.6 ag/mL in a 100-µL PSA solution, 0.97 aM in terms of molar concentration. The present temperature-responsive liposome-linked immunosorbent assay provides an advanced platform for the rapid and highly sensitive detection of biomarkers for use in diagnosis and biological inspections.

Innovations in Extractive Phases for In-Tube Solid-Phase Microextraction Coupled to Miniaturized Liquid Chromatography: A Critical Review.

Over the past years, a great effort has been devoted to the development of new sorbents that can be used to pack or to coat extractive capillaries for in-tube solid-phase microextraction (IT-SPME). Many of those efforts have been focused on the preparation of capillaries for miniaturized liquid chromatography (LC) due to the reduced availability of capillary columns with appropriate dimensions for this kind of system. Moreover, many of the extractive capillaries that have been used for IT-SPME so far are segments of open columns from the gas chromatography (GC) field, but the phase nature and dimensions are very limited. In particular, polar compounds barely interact with stationary GC phases. Capillary GC columns may also be unsuitable when highly selective extractions are needed. In this work, we provide an overview of the extractive capillaries that have been specifically developed for capillary LC (capLC) and nano LC (nanoLC) to enhance the overall performance of the IT-SPME, the chromatographic separation, and the detection. Different monolithic polymers, such as silica C18 and C8 polymers, molecularly imprinted polymers (MIPs), polymers functionalized with antibodies, and polymers reinforced with different types of carbon nanotubes, metal, and metal oxide nanoparticles (including magnetic nanoparticles), and restricted access materials (RAMs) will be presented and critically discussed.

A Lipid-Nanopillar-Array-Based Immunosorbent Assay.

Since infectious diseases, particularly viral infections, have threatened human health and caused huge economical losses globally, a rapid, sensitive, and selective virus detection platform is highly demanded. Enzyme-linked immunosorbent assay (ELISA) with flat solid substrates has been dominantly used in detecting whole viruses for its straightforwardness and simplicity in assay protocols, but it often suffers from limited sensitivity, poor quantification range, and a time-consuming assay procedure. Here, a lipid-nanopillar-array-based immunosorbent assay (LNAIA) is developed with a nanopillar-supported lipid bilayer substrate with fluorophore-modified antibodies for rapid, sensitive, and quantitative detection of viruses. 3D nanopillar array structures and fluid antibodies with fluorophores facilitate faster and efficient target binding and rapid fluorophore localization for quick, reliable analysis on binding events with a conventional fluorescence microscopy setup. LNAIA enables quantification of H1N1 virus that targets down to 150 virus particles with 5-orders-of-magnitude dynamic range within 25 min, which cannot be achieved with conventional ELISA platforms.

Fiber optic nanogold-linked immunosorbent assay for rapid detection of procalcitonin at femtomolar concentration level.

A rapid and ultrasensitive biosensing method based on fiber optic nanogold-linked immunosorbent assay is reported. The method employs an immobilized capture probe on the fiber core surface of an optical fiber and a detection probe conjugated to gold nanoparticles (AuNPs) in a solution. Introduction of a sample containing an analyte and the detection probe into a biosensor chip leads to the formation of a sandwich-like complex of capture probe-analyte-detection probe on the fiber core surface, through which nanoplasmonic absorption of the fiber optic evanescent wave occurs. The performance of this method has been evaluated by its application to the detection of procalcitonin (PCT), an important biomarker for sepsis. In this study, anti-PCT capture antibody is functionalized on an unclad segment of an optical fiber to yield a fiber sensor and anti-PCT detection antibody is conjugated to AuNPs to afford nanoplasmonic probes. The method provides a wide linear response range from 1 pg/mL to 100 ng/mL (5 orders) and an extremely low limit of detection of 95 fg/mL (7.3 fM) for PCT. In addition, the method shows a good correlation in determining PCT in blood plasma with the clinically validated electrochemiluninescent immunoassay. Furthermore, the method is quick (analysis time ≤15 min), requires low-cost instrumentation and sensor chips, and is also potentially applicable to the detection of many other biomarkers.

Highly sensitive fluorescence-linked immunosorbent assay based on aggregation-induced emission luminogens incorporated nanobeads.

Aggregation-induced emission luminogens (AIEgens) have attracted considerable interest for application towards the development of various biosensors due to their unique optical properties. However, the major challenge associated with generating a suitable fluorescent signal for constructing an AIEgens-based immunoassay platform, is the complex surface modification and additional chemical reaction required to activate the AIE process. This work reports a novel AIEgens nanobeads-based fluorescence-linked immunosorbent assay (FLISA) platform wherein the fluorescent labels are hexaphenylsilole (HPS) nanobeads, which were synthesized through Shirasu porous glass (SPG) membrane emulsification method and could provide a strong, direct fluorescent signal without any pretreatment. Moreover, the particle-based signal amplification effect affords this platform significantly improved detection sensitivity for carcinoembryonic antigen (CEA) quantitation. Compared to FLISA which uses R-phycoerythrin (PE) or commercial green QDs nanobeads as fluorescent labels, this AIEgens nanobeads-based FLISA platform exhibits detection sensitivity improved up to 45-fold and 12-fold, respectively. Clinical validation experiments applying this AIEgens nanobeads-based FLISA immunoassay platform to analyze human serum samples produce results consistent with those obtained by the clinical gold-standard method, electrochemiluminescence immunoassay (ECLIA). The strong photobleaching resistance and excellent fluorescent stability of the HPS nanobeads negate the need for light shielding, which improves the efficiency and makes the operating conditions more comfortable. Thus, this AIEgens nanobeads-based FLISA platform, with attractive features including direct fluorescent signal generation and significant signal amplification, creates a new, versatile route for the application of AIEgens in biosensors and clinical diagnosis.

Sensitive and Simple Competitive Biomimetic Nanozyme-Linked Immunosorbent Assay for Colorimetric and Surface-Enhanced Raman Scattering Sensing of Triazophos.

The biomimetic enzyme-linked immunosorbent assay (BELISA) is widely used for detection of small-molecule compounds as a result of low cost and reagent stability of molecularly imprinted polymers (MIPs). However, enzyme labels used in BELISA still suffer some drawbacks, such as high production cost and limited stability. To overcome the drawbacks, a biomimetic nanozyme-linked immunosorbent assay (BNLISA) based on MIPs and nanozyme labels was first proposed. For nanozyme labels, platinum nanoparticles (PtNPs) acted as peroxidase by catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) into an ideal surface-enhanced Raman scattering (SERS) marker. Blue TMB2+ and bovine serum albumin (BSA)-hapten showed superior selectivity when competing with targets for binding sites on MIPs, named the Pt@BSA-hapten probe. The BNLISA method was employed to detect triazophos with a limit of detection of 1 ng mL-1 via colorimetric and SERS methods. Replacing traditional enzymes with nanozymes for combination with MIPs may bring about a new prospect for other compound analyses.

Simultaneous colorimetric determination of acute myocardial infarction biomarkers by integrating self-assembled 3D gold nanovesicles into a multiple immunosorbent assay.

An improved enzyme-free immunosorbent assay is described for the simultaneous detection of the myocardial infarction biomarkers N-terminal pro B type natriuretic peptide (NT-proBNP), creatine kinase-MB (CK-MB), and cardiac muscle troponin T (cTnT). The assay integrates 3D gold nanovesicles (GNVs) and three allochroic agents (phenolphthalein, methyl red, bromothymol blue). The pH regulated allochroic agents were enwrapped in GNVs to acts as ultrasensitive nanoprobes. Loading can be controlled by adjusting the temperature to efficiently load and release the allochroic agents. This bare-eye multicolor assay has limits of detection of 70 pg·mL-1 for NT-proBNP, 910 pg·mL-1 for CK-MB, and 7.8 pg·mL-1 for cTnT. Other features include (a) a linear range that extends over a wide range and sometimes is better than conventional HRP-based immunoassays, and (b) a precision that is comparable to immunofluorescence assays as used in the clinical laboratory. Graphical abstract Schematic presentation of an improved enzyme-free immunosorbent assay (EFISA). It integrates 3D gold nano-vesicles (GNVs) and allochroic agents for the simultaneous detection of acute myocardial infarction (AMI) biomarkers (N-terminal prohormone of brain natriuretic peptide (NT-proBNP), kinase-muscle/brain test (CK-MB), and cardiac muscle troponin (cTnT)).

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti-Citrullinated Protein/Peptide Antibody.

Quantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients' seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious.

Development of immunosorbents for the analysis of forchlorfenuron in fruit juices by ion mobility spectrometry.

The advantages of using smart materials as immunosorbents in the analysis of complex matrices by ion mobility spectrometry (IMS) have been highlighted in this study. A novel analytical method has been proposed for the sensitive, selective, and fast determination of residues of the plant growth regulator forchlorfenuron in fruit juices. Three different monoclonal antibodies (s3#22, p2#21, and p6#41) were employed for the production of immunosorbents, based on Sepharose gel beads, which were characterized in terms of loading capacity, solvent resistance, and repeatability for its use in solid-phase extraction (SPE). Immunosorbents that were prepared with antibody p6#44 provided the best performance, with a loading capacity of 0.97 µg, a 10% (v/v) 2-propanol tolerance, and a reusability of at least eight uses. The SPE procedure involved the use of a column with 0.15 g Sepharose beads, containing 0.5 mg antibody, which was loaded to 20 mL of the sample, washed with 2 mL of water plus 2 mL of 10% (v/v) 2-propanol, and eluted with 2 mL of 2-propanol. The cleaned extract was directly analyzed by IMS, giving a limit of detection of 2 µg L-1 with a relative standard deviation of 7.6%. Trueness was assessed by the analysis of blank grape and kiwifruit juice samples spiked with forchlorfenuron concentrations from 10 to 400 µg L-1, with recoveries from 80 to 115%. The analytical performance of the proposed immunosorbent was compared with conventional extraction and cleanup methods, such as QuEChERS and C18-based SPE, giving the cleanest extracts for accurate determinations of forchlorfenuron by IMS. Graphical abstract ᅟ.

Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen Amplified by Releasing pH Indicator Molecules Entrapped in Mesoporous Silica Nanoparticles.

An enzyme-free titer plate-based colorimetric assay utilizing functionalized mesoporous silica nanoparticles (MSNs) entrapping pH-indicator molecules has been developed. Pores in the silica nanoparticles were functionalized with phenyltrimethyloxysilane so that pH indicator molecules (thymolphthalein or TP in the present case) can be tightly entrapped through π-π conjugation. To detect prostate specific antigen (PSA), the TP-containing MSNs were coated with polyethylenimine (PEI), which favors the attachment of the negatively charged secondary anti-PSA antibody. The entrapped thymolphthalein molecules can be readily released from the pores with a simple addition of alkaline solution. The resultant bifunctional MSNs were used for signal-amplified detection of PSA captured by the primary antibody preimmobilized in the wells of a plate. Our method possesses a wide dynamic range (0.5 to 8000 pg/mL) wherein the adsorption of the bifunctional MSNs obeys a modified Langmuir isotherm. A detection limit (LOD) down to as low as 0.36 pg/mL can be attained. Owing to the size uniformity of the MSNs and the obviation of enzyme molecules employed in the enzyme-linked immunosorbent assay (ELISA), excellent reproducibility (RSD = 1.12%) was achieved. The selective detection of PSA in human serum samples demonstrates the amenability of our method to detect important biomarkers in complex biological samples, whereas the performance of the assay in a titer plate ensures high throughput and obviates the use of expensive instruments. Both of these features are prerequisites for clinical settings wherein a great number of samples need to be analyzed in a timely fashion.

Homogeneous Immunosorbent Assay Based on Single-Particle Enumeration Using Upconversion Nanoparticles for the Sensitive Detection of Cancer Biomarkers.

Prostate-specific antigen (PSA) is an intercellular glycoprotein produced primarily by the prostate gland, which is commonly chosen as the initial target for the early diagnosis of prostate cancer. In this work, we demonstrate a simple yet sensitive sandwich-type single-particle enumeration (SPE) immunoassay for the quantitative detection of PSA in a flow chamber. The design is based on the luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs). The carboxyl group-functionalized UCNPs are conjugated with anti-PSA detection antibodies (Ab1) and serve as the luminescence energy donor, while GNPs are modified with anti-PSA capture antibodies (Ab2) and act as the energy acceptor. In the presence of target antigen (i.e., PSA), the specific immnuoreaction brings the donor and acceptor into close proximity, resulting in quenched luminescence. Through statistical counting of the target-dependent fluorescent particles on the glass slide surface, the quantity of antigens in the solution is accurately determined. The dynamic range for PSA detection in Tris-buffered saline (TBS) is 0-500 pM and the limit-of-detection (LOD) is 1.0 pM, which is much lower than the cutoff level in patients' serum samples. In the serum sample assay, comparable LOD was also achieved (i.e., 2.3 pM). As a consequence, this method will find promising applications for the selective detection of cancer biomarkers in clinical diagnosis.

Magnetic Nanozyme-Linked Immunosorbent Assay for Ultrasensitive Influenza A Virus Detection.

Rapid and sensitive detection of influenza virus is of soaring importance to prevent further spread of infections and adequate clinical treatment. Herein, an ultrasensitive colorimetric assay called magnetic nano(e)zyme-linked immunosorbent assay (MagLISA) is suggested, in which silica-shelled magnetic nanobeads (MagNBs) and gold nanoparticles are combined to monitor influenza A virus up to femtogram per milliliter concentration. Two essential strategies for ultrasensitive sensing are designed, i.e., facile target separation by MagNBs and signal amplification by the enzymelike activity of gold nanozymes (AuNZs). The enzymelike activity was experimentally and computationally evaluated, where the catalyticity of AuNZ was tremendously stronger than that of normal biological enzymes. In the spiked test, a straightforward linearity was presented in the range of 5.0 × 10-15-5.0 × 10-6g·mL-1 in detecting the influenza virus A (New Caledonia/20/1999) (H1N1). The detection limit is up to 5.0 × 10-12 g·mL-1 only by human eyes, as well as up to 44.2 × 10-15 g·mL-1 by a microplate reader, which is the lowest record to monitor influenza virus using enzyme-linked immunosorbent assay-based technology as far as we know. Clinically isolated human serum samples were successfully observed at the detection limit of 2.6 PFU·mL-1. This novel MagLISA demonstrates, therefore, a robust sensing platform possessing the advances of fathomable sample separation, enrichment, ultrasensitive readout, and anti-interference ability may reduce the spread of influenza virus and provide immediate clinical treatment.