Sensitivity Of Antigen Detection Research Articles

Validity of Rapid Diagnostic Test Kit Keeping Light Microscopy in Diagnosing Malaria in Febrile patients

Background: Malaria is the common health problematic disease and it causes major number of mortality and morbidity. It must be diagnosed accurately and rapidly. A rapid and correct diagnosis can reduce the sufferings. So it’s essential to establish an appropriate and reliable laboratory procedure for malaria diagnosis. Microscopic examination of blood film is taken as the gold standard method. Aim: To assess the reliability and validity of rapid diagnostic test kit, so that the rapid and a cost effective diagnostic method may be adopted for the timely diagnosis, and treatment may be offered to patients Methodology: 350 patients having history of fever were taken for this Cross sectional validation study. Tested by ICT rapid diagnostic kit for detection of malarial antigen and results were then compared with gold standard microscopic results Results: for the 350 suspected patients the rate of positivity and negativity were 60% and 40% respectively by microscopic examination and for ICT for detection of malarial antigen were 58% and 42% respectively. Specificity and sensitivity for antigen detection by ICT when it was compared with microscopy of blood smear was 95% and 93.33% respectively. Its overall diagnostic accuracy was good and it was 94% Conclusions: ICT for antigen can diagnose malaria reliably. It is rapid; simple has good sensitivity and specificity. It can detect very low level of parasitic infection and can be performed at point of care and for screening at mass level. No special equipment or high level of setup and skills needed for it. Keywords: Malaria, test kit, microscope

Trimetallic nanoparticle-decorated MXene nanosheets for catalytic electrochemical detection of carcinoembryonic antigen via Exo III-aided dual recycling amplifications

Changes in trace levels of tumor markers are important indicators for diagnosing and treating different diseases, including cancers. On account of a recycled dual amplification approach aided by exonuclease III (Exo III) and the trimetallic nanoparticle-decorated MXene nanosheet (Au-Pd-Pt/Ti3C2Tx)-modified electrode as the catalytic sensing interface, we describe the establishment of a label-free and aptamer-based sensitive carcinoembryonic antigen (CEA) detection assay platform. The target CEA molecules bind the aptamer containing hairpin probes to trigger cyclic cleavage of the secondary hairpins with the assistance of Exo III to release a large amount of ssDNAs, which further hybridize with the G-quadruplex-integrated triple-helix complex (THC) signal probes on the sensor electrode. Exo III then cyclically cleaves the resulting duplexes to liberate many G-quadruplex sequences that can confine hemin on the electrode surface. Subsequent catalytic reduction of H2O2 mediated by hemin on Au-Pd-Pt/Ti3C2Tx-modified sensing interface thus yields drastically enhanced current signals for detecting CEA with high sensitivity between 1 fg mL−1 and 1 ng mL−1 with the detection limit of 0.32 fg mL−1. Such a demonstration of our assay method for CEA indicates that the developed sensing platform can be extended as a promising means for sensitive monitoring of different molecular biomarkers.

A highly sensitive impedimetric sensor based on a MIP biomimetic for the detection of enrofloxacin.

The benefits of molecularly imprinted polymer (MIP) technology in creating artificial receptors to replace natural counterparts have piqued the interest of numerous researchers in recent years. We propose a biomimetic enrofloxacin-MIP for enrofloxacin (ENRO) antigen detection using gold nanoparticles (AuNPs) and MIP methodologies in this study. A self-assembled monomer layer of aminothiophenol was used to immobilize a pre-formed complex of the anti-enrofloxacin monoclonal antibody and enrofloxacin antigen onto the surface of an AuNP coated screen-printed carbon ink electrode (SPCE). The poly-(aminothiophenol) layer thickness was adjusted to entrap and restrict enrofloxacin antigens near the surface. The imprinting and removal of the enrofloxacin antigen in the MIP film were strongly validated by the Raman spectra. The final mAb-MIP sensor had better sensitivity (302 Ω mL ng-1) and a better detection limit (0.05 ng mL-1) than self-assembled monolayer (SAM)-based immunosensors, which had 102 Ω mL ng-1 and 0.1 ng mL-1, respectively.

Rapid and sensitive multiplex detection of COVID-19 antigens and antibody using electrochemical immunosensor-/aptasensor-enabled biochips.

We report protein- and aptamer-based electrochemical biochips for low-cost, one-step, sensitive and accurate multiplex detection of SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and IgG antibody in unprocessed clinical samples, allowing citizens to achieve rapid diagnosis at home or in community settings.

Clinical Trial: Magnetoplasmonic ELISA for Urine-based Active Tuberculosis Detection and Anti-Tuberculosis Therapy Monitoring.

The coronavirus disease 2019 (COVID-19) pandemic has proved the importance of fast and widespread diagnostic testing to prevent serious epidemics timely. The first-line weapon against rapidly transmitted disease is a quick and massive screening test to isolate patients immediately, preventing dissemination. Here, we described magnetoplasmonic nanozymes (MagPlas NZs), i.e., hierarchically coassembled Fe3O4–Au superparticles, that are capable of integrating magnetic enrichment and catalytic amplification, thereby the assay can be streamlined amenable to high-throughput operation and achieve ultrahigh sensitivity. Combining this advantage with conventional enzyme-linked immunosorbent assay (ELISA), we propose a MagPlas ELISA for urine-based tuberculosis (TB) diagnosis and anti-TB therapy monitoring, which enables fast (<3 h), and highly sensitive (up to pM with naked-eyes, < 10 fM with plate reader) urinary TB antigen detection. A clinical study with a total of 297 urine samples showed robust sensitivity for pulmonary tuberculosis (85.0%) and extra-pulmonary tuberculosis (52.8%) patients with high specificity (96.7% and 96.9%). Furthermore, this methodology offers a great promise of noninvasive therapeutic response monitoring, which is impracticable in the gold-standard culture method. The MagPlas ELISA showed high sensitivity comparable to the PCR assay while retaining a simple and cheap ELISA concept, thus it could be a promising point-of-care test for TB epidemic control and possibly applied to other acute infections.

Surface-enhanced Raman scattering of CoV-SARS-2 viral proteins in a strong coupling regime

Fast, sensitive, high-throughput detection of coronavirus antigens at physiologically relevant levels is essential for population screening that could prevent epidemics such as the current COVID-19 global pandemic. Optical methods based on surface-enhanced Raman scattering (SERS) spectroscopy are promising for this purpose because they ensure quick detection of even single biological molecules in a sample. For achieving such a high sensitivity, it is crucial to design SERS-active systems concentrating incident radiation into small sample volumes. Here, metal-dielectric cavities have been obtained through interaction of protein sulfhydryl groups with a SERS-active silver surface. The concentration of light in these cavities allows the differential detection of spike glycoprotein and nucleocapsid protein of SARS-COV-2, which are its key antigens, at physiologically relevant concentrations. The cavity Q-factor can be increased by additionally covering the dielectric protein film with a silver shell to form an ultrathin cavity, which provides an at least tenfold enhancement of the detection signal. The results could be used to design high-throughput systems for specific and sensitive detection of viral antigens and quick diagnosis of viral infections.

Development of highly sensitive and rapid antigen detection assay for diagnosis of COVID-19 utilizing optical waveguide immunosensor.

Development of highly sensitive and rapid antigen detection assay for diagnosis of COVID-19 utilizing optical waveguide immunosensor.

Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles.

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

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.

A novel sandwich-type SERS immunosensor for selective and sensitive carcinoembryonic antigen (CEA) detection

Monitoring the malignant tumors via cancer biomarkers is very significant process. Nonetheless, the practical clinical applications need selective and sensitive analytical methods/techniques. In this study, a novel sandwich type immunosensor based on surface-enhanced raman scattering (SERS) was presented including 4-mercaptobenzoic acid labeled MoS2 nanoflowers@Au nanoparticles (MoS2 NFs@Au NPs/ MBA) as CEASERS tag and Fe3O4@Au nanoparticles functionalized delaminated Ti3C2Tx MXene (Fe3O4 NPs@Au NPs/d-Ti3C2TX MXene) as SERS magnetic supporting substrate for carcinoembryonic antigen (CEA) detection. Especially, the determination of single molecule by using SERS method enables early diagnosis of major diseases. In addition, this technique can be utilized for multiplex analyzes owing to narrow well-resolved peaks. The prepared CEASERS tag and SERS magnetic supporting substrate were characterized by scanning electron microscope (SEM), x-ray diffraction (XRD) method, x-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR). A linearity of 0.0001–100.0 ng mL−1 was observed with high sensitivity. Finally, sandwich type immunosensor demonstrated good selectivity and stability for target CEA recognition in plasma sample.

Advances in Diagnosis of Progressive Pulmonary and Disseminated Coccidioidomycosis.

BackgroundAntibody detection is the main method for diagnosis of coccidioidomycosis, but it has limitations. The Coccidioides antigen enzyme immunoassay is recommended for testing cerebrospinal fluid in suspected meningitis. Reports on urine and serum antigen detection evaluated small numbers of patients who were mostly immunocompromised. The purpose of this study was to assess the accuracy of combined antibody and antigen detection for diagnosis.MethodsA retrospective study, including all patients in whom Coccidioides antigen detection in serum was performed between January 2013 and May 2017, was conducted at Valleywise Health Medical Center (formerly Maricopa Integrated Health System). Sensitivity and specificity of antigen and antibody were evaluated in 158 cases and 487 controls.ResultsThe sensitivity of antibody detection by immunodiffusion (ID) was 84.2%. The sensitivity of antigen detection was 57.0% if both urine and serum were tested and 36.7% if urine alone was tested. The sensitivity of combining antigen and ID antibody detection was 93.0%. The sensitivity of urine and serum antigen detection was 55.4% in proven and 58.7% in probable cases, 79.1% in disseminated and 41.6% in pulmonary cases, and 74.7% in immunocompromised and 40.0% in immunocompetent patients. Specificity was 99.4% for antigen detection and 96.5% for ID antibody detection. Diagnostic accuracy was 95.4% for ID antibody and antigen detection, 93.6% for ID antibody alone, and 89.1% for pathology or culture.ConclusionsThese findings support combined antibody and antigen detection for diagnosis of progressive coccidioidomycosis. The diagnosis may have been missed if antigen detection was not performed.

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

Surface-enhanced electrochemiluminescence combined with resonance energy transfer for sensitive carcinoembryonic antigen detection in exhaled breath condensates.

The detection of biomarkers in exhaled breath condensates (EBCs) is regarded as a promising non-invasive diagnostic approach. However, the ultralow concentration of biomarkers in EBCs is a great challenge. Herein, a sensitive dual signal amplification strategy was developed based on surface-enhanced electrochemiluminescence (SEECL) combined with resonance energy transfer (RET). Gold nanoparticles-functionalized graphite-like carbon nitride nanohybrids (Au-g-C3N4 NHs) could be used as an energy transfer donor because of the good overlap between its emission peak and the absorption peak of tris(2,2'-bipyridine)ruthenium dichloride (Ru(bpy)3Cl2) at 460 nm. Gold-silicon dioxide core-shell nanoparticles doped with Ru(bpy)32+(Au@SiO2-Ru) were employed as an energy transfer acceptor emitting at 620 nm. Moreover, the signals at 620 nm emitted by Ru (bpy)32+ were enhanced by 5 times, attributed to the localized surface plasmon resonance (LSPR) of gold nanoparticles (Au NPs). The detection of carcinoembryonic antigen (CEA) was performed by using two aptamers as the recognition unit; whereby aptamer 1 (Apt1) was modified on the surface of Au-g-C3N4 NHs, and aptamer 2 (Apt2) was banded on the surface of Au@SiO2-Ru. In the presence of CEA, a sandwich structure was formed between Au-g-C3N4 NHs-Apt1-CEA and Apt2-Au@SiO2-Ru, which resulted in an ultrasensitive detection of CEA. The proposed electrochemiluminescence sensor showed a wide linear relationship with the CEA concentration in the range from 1.0 pg mL-1 to 5.0 ng mL-1, with a limit of detection (LOD) of 0.3 pg mL-1. Finally, the practicality of the proposed sensor was demonstrated to detect CEA in EBCs, and the obtained results were in good agreement with the enzyme-linked immunosorbent assay (ELISA) method.

263. Advances in Diagnosis of Progressive Coccidioidomycos: Experience in 164 Cases and 508 Controls

BackgroundAntibody detection is the main method for diagnosis of coccidioidomycosis but has limitations including sensitivity and turnaround time. The MVISTA Coccidioides antigen enzyme immunoassay (EIA) is recommended for testing CSF in suspected Coccidioides meningitis. The early reports on urine and serum antigen testing evaluated small numbers of patients who were mostly immunocompromised with advanced disease.MethodsA retrospective study, including all patients in whom Coccidioides antigen testing was performed between January 2013 and May 2017, was conducted at Maricopa Integrated Health System (MIHS). Sensitivity and specificity of antigen testing at MiraVista Diagnostics and antibody testing at MIHS or commercial laboratories were evaluated in 164 cases and 508 controls.ResultsThe sensitivity of antigen testing was 51% and specificity was 99%. The sensitivity of antigen detection was highest if both urine and serum were tested (57%) than if only urine was tested (38%). The sensitivity of antibody testing was 84% and the specificity was 94% by immunodiffusion (ID). The sensitivity and specificity of antigen or ID antibody testing both were 94%. Sensitivity of antigen testing was 57% in proven and 58% in probable cases, ID antibody in 85% of proven and 75% of probable and antigen or ID antibody in 93% of proven and 95% of probable cases. Antigen was detected more often in disseminated (79%) than pulmonary cases (42%) as was ID antibody, 91% and 79%, respectively. Antigen testing was more sensitive in immunocompromised (76%) than non-immunocompromised patients (41%) while ID antibody was less sensitive in immunocompromised (74%) than in non-immunocompromised patients (93%). Combined antigen and ID antibody testing provided the highest sensitivity, 94% in all cases, 94% in immunocompromised and 95% in non-immunocompromised patients.ConclusionThese findings support testing urine and serum for Coccidioides antigen and serum for ID antibodies for diagnosis of progressive pulmonary or disseminated coccidioidomycosis. Disclosures All authors: No reported disclosures.

Echinococcus granulosus protoscolex antigen used in serodiagnosis of hydatidosis by nano-gold dot-ELISA

Background: Echinococcus granulosus protoscolex antigen (PSAg) is a protein with significant immunological properties having higher sensitivity and specificity in ELISA. It lowers cross-reaction with antibodies of other parasites and thus its application is recommended in serological diagnosis. Labelling of ELISA with nano-gold particles improved the diagnostic abilities of the laboratory technique in hydatidosis detection. Objective: To evaluate the use of nano-gold dot-ELISA for isolation of E. granulosus PSAg and its application in serodiagnosis of hydatidosis in humans and animals in comparison with dot-ELISA. Material and Methods: Hydatid cyst PSAg was isolated and used for immunization of rabbits to raise IgG polyclonal antibodies (pAb) in antisera. These sera were labeled with horseradish peroxidase (HRP) and used for detection of circulating PSAg in sera of human cases and camels and sheep by dot-ELISA and nano-gold dot-ELISA. Results: Conjugation of the anti-protoscolex pAb with gold nano-particles increased the sensitivity of antigen detection by nano-gold dot-ELISA to 94.4% and specificity to 90%, with positive and negative predictive values of 94.4% and 90%, and an accuracy of detection of 92.9% in both human and animal sera. Conclusion: Nano-gold dot-ELISA technique is more sensitive than dot-ELISA for detection of hydatidosis antigen both in human and animal samples

Cu-DNAzyme facilitates highly sensitive immunoassay

Fast and sensitive antigen detection is important in biomedical research and development. Despite, being invented 48 years ago, the enzyme-linked immunosorbent assay (ELISA) remains one of the most successful and widely employed bioanalytical techniques in research and clinical diagnostics due to its reliability and simplistic design. Recently, nanotechnology has offered efficient signal reporting. In spite of some improvements in these systems, there are typically increased material costs involved and the need for expensive equipment or complicated chemical processes, thus negating any possible benefits over ELISA. Herein, we communicate a simple Cu-DNAzyme system for signal transduction of a CuO nanoparticle-labeled immunoassay. The reported immunoassay amplifies signal generation similar to traditional ELISA and is fast, simple, cost-effective, and sensitive, holding promise for biomedical applications and point-of-care testing.

Enhanced Visual Wireless Electrochemiluminescence Immunosensing of Prostate-Specific Antigen Based on the Luminol Loaded into MIL-53(Fe)-NH2 Accelerator and Hydrogen Evolution Reaction Mediation.

A sensitive prostate-specific antigen (PSA) detection method using a visual-readout closed bipolar electrode (BPE) system has been introduced by integration of hydrogen evolution reaction (HER) in cathodic pole and electrochemiluminescence (ECL) of luminol loaded within the MIL-53(Fe)-NH2 (L@MIL-53(Fe)-NH2) in the anodic pole. The cathode of the BPE was electrochemically synthesized by 3D porous copper foam, followed by decorating with nitrogen-doped graphene nanosheet and ruthenium nanoparticles. As an alternative, we employed carboxylate-modified magnetic nanoparticles (MNPs) for immobilization of the primary antibody (Ab1) and utilized the L@MIL-53(Fe)-NH2 conjugated to secondary antibody (Ab2) as a signaling probe and coreaction accelerator. After sandwiching the target PSA between Ab1 and Ab2, the MNP/Ab1-PSA-Ab2/L@MIL-53(Fe) were introduced to a gold anodic BPE. Finally, the resulting ECL of luminol and H2O2 at the anodic poles was monitored using a photomultiplier tube (PMT) or digital camera. The PMT and visual (camera)-based detections showed linear responses from 1 pg mL-1 to 300 ng mL-1 (limit of detection 0.2 pg mL-1) and 5 pg mL-1 to 200 ng mL-1 (limit of detection 0.1 pg mL-1), respectively. This strategy provides an effective method for high-performance bioanalysis and opens a new door toward the development of the highly sensitive and user-friendly device.

Green-emitting carbon dot loaded silica nanoparticles coated with DNA-cross-linked hydrogels for sensitive carcinoembryonic antigen detection and effective targeted cancer therapy.

Herein, we have designed bifunctional composite nanospheres for carcinoembryonic antigen (CEA) sensing and targeted drug delivery, based on carbon dot loaded silica nanoparticles coated with DNA-cross-linked hydrogels. As a result, highly sensitive and selective CEA detection was achieved in vitro, and an effective cytotoxic effect was realized in vivo after loading doxorubicin.

Selective, sensitive and comprehensive detection of immune complex antigens by immune complexome analysis with papain-digestion and elution

Comprehensive identification and profiling of antigens in immune complexes (ICs) in biological fluids, such as serum and cerebrospinal fluid, is useful for developing early diagnostic markers and specific treatments for many diseases. We have developed a method, designated “immune complexome analysis”, to comprehensively identify the antigens in ICs. In this method, we first purify ICs from biological fluid by using Protein G- or Protein A-coated beads, then these ICs are subjected to tryptic digestion on the beads and subsequent analysis using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We previously used this method to find specific antigens in circulating ICs (CIC-antigens) in serum for autoimmune diseases, infectious disease and cancers. However, this method detects not only CIC-antigens but also antibodies and proteins bound non-specifically to the beads, which restricts the detection of minor peptides released by the digestion of CIC-antigens whose amounts are generally much less than antibodies and the proteins. To selectively detect CIC-antigens with enhanced sensitivity, in this study we compared three methods (Method A, direct tryptic digestion on the beads; Method B, low-pH elution and tryptic digestion; Method C, papain-digestion, elution, and tryptic digestion) and examined which method selectively elutes CIC-antigens from CICs bound to the beads and selectively detects CIC-antigens using nano-LC-MS/MS. We also compared three types of CIC-capturing beads (Protein G-coated magnetic beads, Protein A-coated magnetic beads and Proceptor™-sepharose beads) to examine if parallel use of these beads aids the comprehensive detection of CIC-antigens in immune complexome analysis. Comparison showed that Method C provided the most selective and sensitive detection of CIC-antigens, without interference by antibodies and proteins non-specifically bound to the beads. In addition, using three types of beads allowed the examination of a wide range of CIC-antigens in immune complexome analysis. Therefore, combining Method C with three types of beads should allow the selective and sensitive identification of IC-antigens present in biological fluids from patients with a variety of diseases. The identification of IC-antigens may lead to the development of diagnostic methods and protocols for specific treatments for these diseases.

Liposome-amplified photoelectrochemical immunoassay for highly sensitive monitoring of disease biomarkers based on a split-type strategy

Liposomes are an excellent candidate component for biosensors to transduce and amplify detection signals due to their outstanding ability in encapsulating signal marker compounds. However, the use of liposomes for photoelectrochemical (PEC) signal transduction has not yet been achieved due the lack of appropriate sensing strategy. Herein, we report on a novel liposomes-amplified PEC immunoassay (LAPIA) method for sensitive HIV-p24 antigen (p24) detection based on a split-type strategy. Initially, liposomes were encapsulated with alkaline phosphatase (ALP) in their hydrophilic chamber and conjugated with secondary antibody on the surface to form the ALP-encapsulated liposomes (ALP-Ls) based PEC signal label. Sandwiched immunoassay based on the ALP-Ls label was then carried out in microwell plate. Upon addition of tween 20, the ALP molecules were released and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid (AA). The latter then donated electron to the graphene/g-C3N4 nanohybrids based photoelectrode, arousing an increased photocurrent signal. The separation of immunoreaction step and PEC signal excitation (i.e. split-type) not only enabled the realization of liposomes based amplification strategy, but also could eliminate the PEC-caused biomolecules damage. The developed PEC method possessed a wide calibration range from 1.0pgmL−1 to 50ngmL−1 and a low detection limit of 0.63pgmL−1. Its practicability was demonstrated by assaying human serum samples. Moreover, the universality of the liposomes-amplified PEC sensing strategy was also demonstrated by developing it into a sensitive microRNA detection method.