Combined detection of SARS-CoV-2 neutralizing antibodies and specific IgG in plasma based on SERS magnetic sensor.

In this study, a surface-enhanced Raman spectroscopy (SERS) magnetic sensor is established based on SERS principle and magnetic separation technology, and a highly sensitive, simple and fast method for quantitative detection of neutralizing antibodies (nABs) and specific IgG of SARS-CoV-2 in plasma is established combined with immunoassay. Two kinds of Raman nanospheres (RNPs) with different characteristic Raman shifts are used as signal sources and coupled to ACE2 and anti-IgG (FC) antibodies respectively, and magnetic beads are coupled to RBD. The competitive relationship between ACE2 and nABs, the binding relationship between specific IgG and anti-IgG (FC) antibodies are determined. The results show that the concentrations of nABs and specific IgG in the range of 10-2000 ng ml-1are well correlated with SERS response intensity, and the recoveries are both between 90%-110%, with good precision. Bilirubin and common anticoagulants have no interference on the detection results. This method is accurate, reliable, sensitive and does not require complex pre-treatment, and is expected to be used for simultaneous detection of nABs and specific IgG in plasma of SARS-CoV-2. It has guiding significance for the development and evaluation of vaccines and the formulation of individualized vaccination schedule.

Development of a system for detecting cardiac troponin I by background fluorescence quenching based on internal filtration effect.

The present study sought to develop a cardiac troponin I (cTnI) detection system based on background fluorescence quenching of internal filtration effect (IFE) and study the influence of IFE on the sensitivity of cTnI detection. Three nanogold materials were synthesized as fluorescence quenchers, and rhodamine 6 G (R6G) and Cy5 were used as fluorescence probes. Six experimental systems were established to detect cTnI in negative serum test solutions and clinical serum samples. The sensitivity of each system was compared to explore the contribution of IFE to the detection sensitivity of cTnI. When applied to negative serum test solutions, the R6G-nanogold material I system exhibited a superior detection effect for cTnI, with a limit of detection (LOD) of 0.15 ng ml-1. When applied to clinical serum samples, the Cy5-nanogold material Ⅲ system yielded a better detection effect for cTnI, with the lowest concentration of cTnI detected at 2 ng ml-1. The first and second internal filtering effects in the proposed system can be achieved simultaneously, effectively avoiding light absorption interference from clinical serum samples and enhancing the sensitivity of the background fluorescence quenching detection of cTnI.

Study on the performance of novel nanomaterials for detection of biomarkers such as PCT based on immunochromatography sensitivity.

In this study, by comparing the UV-vis spectral characteristics of colloidal gold and colloidal gold enhancer, and their differences as immunochromatographic tracers in the qualitative detection of PCT, IL-6, Hp and quantitative determination of PCT performance, the factors that may affect the sensitivity were discussed. The results show that the absorbance at 520 nm of CGE diluted 20-fold and colloidal gold diluted 2-fold were comparable, and the sensitivity of CGE immunoprobe for qualitative detection of PCT, IL-6 and Hp was higher than that of colloidal gold immunoprobe, and the reproducibility and accuracy of both immunoprobes for quantitative detection of PCT were good. Indicating that the high sensitivity of CGE immunoprobe detection is mainly due to the absorption coefficient of CGE at 520 nm is about 10 times that of colloidal gold immunoprobe, CGE has stronger light absorption capacity and stronger quenching effect on rhodamine 6 G on the nitrocellulose membrane surface of the test strip.

Rapid and sensitive detection of amphetamine by SERS-based competitive immunoassay coupled with magnetic separation.

Amphetamine (AMP), as a psychiatric drug acting on the central nervous system, and has become one of the most common drugs of abuse in the illegal market at present, which adversely affects social public safety. We developed a SERS magnetic immunoassay with high sensitivity, specificity, and rapid and quantitative detection of AMP. We synthesized a high SERS intensity substrate (Au-XP013@Ag) using the "hot spot" effect and combined it with antibodies to form SERS immunotags (Au-XP013@Ag-AMP-mAb). Subsequently, the carboxyl magnetic beads were linked to label antigens as functional magnetic beads (carboxyl magnetic beads-AMP-BSA). Using the principle of competitive immunoassay, the Raman response value of the immune complex formed with SERS tags and functional magnetic beads was detected to realize the quantitative detection of AMP. The detection limit of this method for AMP was 2.28 ng mL-1. More importantly, a portable Raman instrument was used in this study, which can meet the requirements of point-of-care testing (POCT). Therefore, this SERS-based magnetic immunoassay can provide a favorable scientific basis for the control of drug abuse, monitoring by law enforcement agencies, and determination of drug users.

Synchronous detection of IgG subtypes based on SERS combined with immunoassay.

In this study, a rapid, simple, highly sensitive and anti-interference method for the joint detection of four IgG subtypes is established by using Raman microspheres with four characteristic Raman spectra. The results show that the concentrations of IgG1 in the range of 0-1500 ng ml-1, IgG2 in the range of 0-1100 ng ml-1, IgG3 in the range of 0-88.7 ng ml-1, IgG4 in the range of 0-77.2 ng ml-1, it shows a good correlation with the response value of The Raman signal. The lowest detection limits are 25.4 ng ml-1, 21.7 ng ml-1, 1.6 ng ml-1, 1.7 ng ml-1, respectively. Reproducibility is good, the coefficient of variation of low, medium and high concentration standard solution are within 10%. The recoveries of four IgG subtypes are in the range of 90%-110%, and the accuracy of the method is good. The coefficients of variation between and within the three batches of reagents are all less than 11%, showing good precision. There is no cross reaction with Procalcitonin (20 ng ml-1), Interleukin-6 (1 ng ml-1) and bovine serum albumin (10 mg ml-1), and the specificity is good. Common interfering substances such as bilirubin, triglyceride and trisodium citrate do not affect the determination results, and heparin sodium only affects the determination results of IgG1. This method has good anti-interference ability. The method has high sensitivity, simple operation and strong anti-interference ability, and has good correlation with the IgG detection methods commonly used in clinic. This simple and quantitative method can be used for the rapid detection of IgG subtypes in the future, which can improve the efficiency of clinical diagnosis.

SERS-based magnetic immunoassay for simultaneous detection of cTnI and H-FABP using core-shell nanotags.

Acute myocardial infarction (AMI) is the single leading cause of worldwide mortality and morbidity. Heart-type fatty acid-binding protein (H-FABP) and cardiac troponin I (cTnI), as biomarkers emerging at different stages of AMI, have complementary advantages in terms of specificity and sensitivity. Therefore, we developed a magnetic immunoassay method based on surface-enhanced Raman scattering (SERS) to detect H-FABP and cTnI simultaneously. Herein, two mutually independent Raman reporter molecules were embedded between a gold core and silver shell and then combined with a tracer antibody to form a SERS immunoprobe. During detection, the SERS immunoprobe, target antigen and capture probe undergo an immune reaction to form a sandwich structure, and then the immune complex was enriched by a specific reaction of streptavidin on the surface of magnetic beads with biotin. Finally, the concentration of biomarkers was quantified by detecting the characteristic Raman peak intensities of the two Raman reporter molecules. Under the optimized conditions, the minimum detection limits of H-FABP and cTnI were 0.6396 and 0.0044 ng mL-1, respectively. Besides, the target antigen does not cross-react with non-specific proteins, showing good specificity. Therefore, our proposed SERS-based magnetic immunoassay method has the advantages of accuracy, rapidity and good selectivity, and has great potential for early diagnosis of acute myocardial infarction disease.

A novel surface-enhanced Raman scattering method for simultaneous detection of ketamine and amphetamine.

As common psychotropic drugs, ketamine (KET) and amphetamine (AMP) are often consumed by drug users at the same time, which seriously threatens people's health. Therefore, the study of simultaneous detection methods for KET and AMP is imperative. In this study, a novel method for the simultaneous detection of KET and AMP in serum was established on the basis of surface-enhanced Raman scattering (SERS). The antibodies were attached on Au@Ag core-shell nanoparticles embedded with different Raman reporters as the detection substrates. The labelled antigens KET-BSA and AMP-BSA were linked to carboxyl magnetic beads, and by adopting the principle of competitive immunoassay, the quantitative detections of ketamine and amphetamine were realized at the same time by detecting the Raman signals at different characteristic peaks on the magnetic beads. A good correlation was shown between ketamine and amphetamine and Raman signal response values were in the concentration range of 0-60 ng mL-1 and 0-200 ng mL-1, and the limits of detection were 1.64 and 2.44 ng mL-1. This method had the advantages of simple, rapid operation, and high sensitivity, and can realise double indicator simultaneous detection, which provided a more favorable scientific basis for preventing or reducing drug abuse, and identifying and monitoring drug users.

Detection of H-FABPA by novel SERS combined with magnetic reaction.

The purpose of this paper is to establish a method for easy operation, high sensitivity, strong anti-interference ability, and rapid quantitative detection of cardiac fatty acid-binding protein in acute myocardial infarction biomarkers, so that it can be quickly diagnosed at an early stage and provide a basis for further treatment. Based on the SERS principle, the traditional sandwich system generated by the reaction was captured by the streptavidin (SA) magnetic beads through the specific reaction of SA and biotin then enriched by the applied magnetic field. The enriched magnetic beads are subjected to Raman detection to achieve a process of quantitative detection of the antigen. The minimum detection limit of this study was 1.4490 ng ml-1, the recoveries were 97.36%-98.35%, and the relative standard deviations between batches and batches were less than 15%. There was no crossover between cTnI, D-dimmer and NT-proBNP. In addition to hemoglobin, the common interfering substances in serum and common anticoagulants do not interfere with the test results. Surface-enhanced Raman spectroscopy can quickly and accurately quantify the acute myocardial infarction marker H-FABP, which is easy to operate and strong in anti-interference ability.

Development of a Quantitative Detection Card for Heart-type Fatty Acid-binding Protein based on Background Fluorescence Quenching Immune Chromatography.

BACKGROUND: To establish a fast and simple quantitative method for detection of heart-type fatty acid-binding protein (H-FABP) in serum based on a background fluorescence quenching immunochromatographic assay. METHODS: A detection card based on the double-antibody sandwich double-antibody method with background fluorescence quenching was developed for quantitative measurement of H-FABP in serum. The optimal concentrations of control for coating the test and control lines were determined as well as the concentrations of gold-labeled antibodies used in preparing the detection system. The detection method for H-FABP in serum was established and validated using real-world clinical samples. RESULTS: The optimal concentrations of labeling antibody and coating antibody were 5.0 µg/mL and 1.0 mg/mL, respectively. The test card had a sensitivity of 1.15 ng/mL over a linear concentration range of 0-100 ng/mL. Based on three batches prepared for testing the card, the relative standard deviation (RSD) within batches was less than 15% without a significant difference (P=0.942). The detection method was tested against common interfering substances in serum, such as bilirubin, triglyceride and serum anticoagulants ethylenediamine tetraacetic acid (EDTA), heparin, and sodium citrate, and no significant cross-reaction was detected. The test method was further validated with 50 clinical serum samples, and the test results were comparable with standard reference detection methods with good correlation (R=0.95). CONCLUSION: Our study presents a new method with strong specificity and sensitivity for the detection of H-FABP in serum, which could promote H-FABP detection in a broad range of applications.

Determination of Aflatoxin M1 and Chloramphenicol in Milk Based on Background Fluorescence Quenching Immunochromatographic Assay.

Harsh demanding has been exposed on the concentration of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. In this study, we developed a new method based on background fluorescence quenching immunochromatographic assay (bFQICA) to detect AFM1 and CAP in milk. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards (i.e., 0.25 ng/mL, 0.5 ng/mL, and 1.0 ng/mL), and the actual detection value was 0.2497, 0.5329, and 1.0941, respectively. For the assay variability of 3 CAP standards (i.e., 0.10 ng/mL, 0.30 ng/mL, and 0.50 ng/mL), the actual detection value was 0.0996, 0.3096, and 0.4905, respectively. The recovery rate of AFM1 was 99.7%-101.7%, while that for CAP was 95.3%-97.6%. For the test stability, AFM1 and CAP showed satisfactory test stability even at month 5. Compared with the sensitivity of liquid chromatography-mass spectrometry (LC-MS) method, no statistical difference was noticed in results of the bFQICA. Our method is convenient for the detection of AFM1 and CAP in milk with a test duration of about 8 minutes. Additionally, an internal WiFi facility is provided in the system allowing for quick connection and storage in the intelligent cell phone.

Antigen detection based on background fluorescence quenching immunochromatographic assay.

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100ngmL(-1) of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1ngmL(-1). Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r=0.98, p<0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases.

Evaluation of a method for the simultaneous detection of multiple tumor markers using a multiplex suspension bead array.

OBJECTIVE: To achieve higher tumor detection efficiency, we evaluated a multiplex assay for TM analysis based on the Luminex-100 multiplex suspension bead array. DESIGN: The assay simultaneously determined the concentrations of nine TMs in 1114 human serum specimens (546 patients with tumors, 158 patients with non-tumor inflammatory diseases, and 410 normal controls). The nine TMs were AFP, CEA, CA125, CYFRA 21-1, CA242, f-PSA, t-PSA, NSE and free ß-hCG. The multiplex suspension bead assays were compared with conventional methods used in clinical laboratories. RESULTS: The Luminex assay has the same levels of sensitivity, specificity and accuracy in the prediction of positive tumor specimens as conventional methods. CONCLUSION: Multiplex suspension bead arrays have promising applications in clinical laboratories.