PEI-Mediated Assembly of Fe3O4 onto SiO2-Encapsulated CsPbBr3 for Highly Sensitive Fluorescent Lateral Flow Immunoassay.

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method's sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL-1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL-1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL-1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation.

A chemiluminescence immunoassay for type IV collagen as a promising marker for liver fibrosis and cirrhosis.

Herein, a magnetic bead-based chemiluminescence assay is reported to detect type IV collagen (col-IV) in serum samples. Magnetic beads (MBs) exhibit biocompatibility. Taking advantage of this property, they were conjugated with the col-IV antibody. For the determination of col-IV, the interaction of the col-IV sample, anti-(col-IV)-alkaline phosphatase (anti-(col-IV)-ALP) and anti-col-IV-magnetic beads (anti-(col-IV)-MBs) was performed to generate chemiluminescence. Under the optimized conditions, the developed method displayed good linearity in the concentration range of 20-2000 ng mL-1 with the limit of 0.79 ng mL-1. The repeatability coefficient of variation (CV) for col-IV detection ranged from 3.16% to 7.50%. The col-IV level in samples collected from a hospital was assessed by the chemiluminescence assay. Satisfactory recoveries were obtained ranging from 93.30% to 100.14%. In conclusion, the magnetic bead-based chemiluminescence assay may be used as a routine and efficient tool to detect type IV collagen in clinical diagnosis.

Simultaneous determination of inflammatory factors SAA and LTF based on stable element labeling and inductively coupled plasma mass spectrometry to aid in the diagnosis of infection.

OBJECTIVE: The clinical value of Serum amyloid A (SAA) and Lactoferrin (LTF) has received significant attention, but their detection methods are inadequate, which limits their application. This study aims to develop a dual detection method based on stable element labeling strategies and inductively coupled plasma mass spectrometry (ICP-MS) for SAA/LTF and to assess whether it can be widely used in clinical practice. METHODS: A duplex immunoassay system based on sandwich method was constructed. After optimization, methodological evaluation was performed with the guidelines of Clinical Laboratory Standards Institute (CLSI). Finally, 131 plasma samples were collected to analyze whether the new method is suitable for clinical detection. RESULTS: The LoB, LLoQ, ULoQ, and linear range of the assay were 1.09 ng/mL, 3 ng/mL, 1500 ng/mL, 3-1500 ng/mL for SAA and 0.85 ng/mL, 2 ng/mL, 1200 ng/mL, 2-1200 ng/mL for LTF respectively. The recovery rates were 95.01% to 106.26%, the intra-batch precision of low, intermediate, and high-level samples was <8%, and the inter-batch of them was <11%, the deviation of interference test results was less than±10%. The Area Under the Curve (AUC) was 0.9809 for SAA, 0.8599 for LTF, and 0.9986 for combination. CONCLUSION: The quantitative duplex immunoassay for SAA/LTF has high accuracy, good precision, and high specificity, which meets the clinical testing requirements and can be widely used in clinical practice.

Plasmonic Annular Nanotrenches with 1 nm Nanogaps for Detection of SARS-CoV-2 Using SERS-Based Immunoassay.

This study represents the synthesis of a novel class of nanoparticles denoted as annular Au nanotrenches (AANTs). AANTs are engineered to possess embedded, narrow circular nanogaps with dimensions of approximately 1 nm, facilitating near-field focusing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via a surface-enhanced Raman scattering (SERS)-based immunoassay. Notably, AANTs exhibited an exceedingly low limit of detection (LOD) of 1 fg/mL for SARS-CoV-2 spike glycoproteins, surpassing the commercially available enzyme-linked immunosorbent assay (ELISA) by 6 orders of magnitude (1 ng/mL from ELISA). To assess the real-world applicability, a study was conducted on 50 clinical samples using an SERS-based immunoassay with AANTs. The results revealed a sensitivity of 96% and a selectivity of 100%, demonstrating the significantly enhanced sensing capabilities of the proposed approach in comparison to ELISA and commercial lateral flow assay kits.

Immunoassay Testing of Alpha-Gal Specific Immunoglobulin-E: Data from a National Reference Laboratory.

BACKGROUND: Immunoassay measurements of serum alpha-gal (AG) specific IgE (sIgE) enable antibody detection and quantification with high sensitivity and specificity and are essential for AG syndrome diagnosis and patient management. We here present and analyze results from over 15 000 patient serum samples tested using the ImmunoCAP (Thermo/Phadia) assay. METHODS: AG-sIgE levels and positivity rates were correlated to patient age, gender, geographic location, repeat testing results, sIgE levels to co-tested red meat whole allergen extracts, and Rocky Mountain spotted fever (RMSF) serology performed on a subset of patient samples. RESULTS: Of the tested samples, 36.7% contained detectable (>0.1 KUA/L) AG-sIgE. Antibody levels were higher in patients of older age, in samples submitted from lower midwestern and southern states, and during the June-December period of the year. Specific IgE to co-tested red meat whole allergens showed moderate to strong correlation to AG-sIgE and were of lower levels. Samples with positive RMSF IgG titers (≥1:64) were of overall higher AG-IgE levels. CONCLUSION: Findings are consistent with the role of lone star ticks in AG syndrome pathogenesis. Levels of measured sIgE to AG are higher than co-tested sIgE to red meat whole allergen, consistent with the improved diagnostic performance of component-resolved testing.

Paper-based electrochemical immunosensor for highly sensitive detection of chicken anemia virus.

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming.

Research on the Flow Characteristics and Reaction Mechanisms of Lateral Flow Immunoassay under Non-Uniform Flow.

Lateral flow immunoassay (LFIA) is extensively utilized for point-of-care testing due to its ease of operation, cost-effectiveness, and swift results. This study investigates the flow dynamics and reaction mechanisms in LFIA by developing a three-dimensional model using the Richards equation and porous media transport, and employing numerical simulations through the finite element method. The study delves into the transport and diffusion behaviors of each reaction component in both sandwich LFIA and competitive LFIA under non-uniform flow conditions. Additionally, the impact of various parameters (such as reporter particle concentration, initial capture probe concentrations for the T-line and C-line, and reaction rate constants) on LFIA performance is analyzed. The findings reveal that, in sandwich LFIA, optimizing parameters like increasing reporter particle concentration and initial capture probe concentration for the T-line, as well as adjusting reaction rate constants, can effectively enhance detection sensitivity and broaden the working range. Conversely, in competitive LFIA, the effects are inverse. This model offers valuable insights for the design and enhancement of LFIA assays.

Synthesis of silver­bismuth oxide encapsulated hydrazone functionalized chitosan (AgBi2O3/FCS) nanocomposite for electrochemical sensing of glucose, H2O2 and Escherichia coli O157:H7.

In this work, silver­bismuth oxide encapsulated 1,3,5-triazine-bis(4-methylbenzenesulfonyl)-hydrazone functionalized chitosan (SBO/FCS) nanocomposite was synthesized by a simple hydrothermal method. The amine (-NH2) group was functionalized by the addition of cyanuric acid chloride followed by 4-methylbenzenesulfonol hydrazide. The SBO/FCS has been characterized by FT-IR, X-ray diffraction, XPS, HR-SEM, HR-TEM, AFM, and thermogravimetry (TGA). Under the optimum conditions, the SBO/FCS sensor showed brilliant electrochemical accomplishment for the sensing of glucose and H2O2 by a limit of detection (LOD) of 0.057 µM and 0.006 µM. It also showed linearity for glucose 0.008-4.848 mM and for H2O2 of 0.01-6.848 mM. Similarly, the sensor exhibited a low sensitivity to glucose (32 µA mM-1 cm-2) and a good sensitivity to H2O2 (295 µA mM-1 cm-2). In addition, that the prepared electrode could be used to sense the glucose and H2O2 levels in real samples such as blood serum and HeLa cell lines. The screen printed electrode (SPE) immunosensor could sense the E. coli O157:H7 concurrently and quantitatively with a linear range of 1.0 × 101-1.0 × 109 CFU mL-1 and a LOD of 4 CFU mL-1. Likewise, the immunosensor efficiently detect spiked E. coli O157:H7 in milk, chicken, and pork samples, with recoveries ranging from 89.70 to 104.72 %, demonstrating that the immunosensor was accurate and reliable.

A one-step immunoassay based on switching peptides for diagnosis of porcine epidemic diarrhea virus (PEDV) using screened Fv-antibodies.

In this study, a one-step immunoassay for porcine epidemic diarrhea virus (PEDV) based on Fv-antibodies and switching peptides was developed, and the assay results of PEDV were obtained by just mixing samples without any further reaction or washing steps. The Fv-antibodies with binding affinity to the spike protein of PEDV were screened from the Fv-antibody library using the receptor-binding domain (RBD) of the spike protein as a screening probe. Screened Fv-antibodies with binding affinities to the RBD antigen were expressed, and the binding constants (KD) were calculated to be 83-142 nM. The one-step immunoassay for the detection of PEDV was configured as a displacement immunoassay using a fluorescence-labeled switching peptide. The one-step immunoassay based on switching peptides was performed using PEDV, and the limit of detection (LOD) values for PEDV detection were estimated to be Ct = 39.7-36.4. Compared with the LOD value for a conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay showed a remarkably improved LOD for the detection of PEDV. Finally, the interaction between the screened Fv-antibodies and the PEDV RBD was investigated using docking simulations and compared with the amino acid sequences of the receptors on host cells, such as aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).

High-Density Au Anchored to Ti3C2-Based Colorimetric-Fluorescence Dual-Mode Lateral Flow Immunoassay for All-Domain-Enhanced Performance and Signal Intercalibration.

In this work, a novel MXene-Au nanoparticle (Ti3C2@Au) was synthesized with a high molar extinction coefficient, strong fluorescence quenching ability, ultrahigh antibody affinity, high stability, and good dispersibility, and it was used to develop a colorimetric-fluorescence dual-mode lateral flow immunoassay (LFIA). The detection limits of this method for the detection of dexamethasone in milk, beef, and pork were 0.0018, 0.12, and 0.084 µg/kg in the "turn-off" mode (colorimetric signal), and 0.0013, 0.080, and 0.070 µg/kg in the "turn-on" mode (fluorescent signal), respectively, which was up to 231-fold more sensitive compared with that of the reported LFIAs. The recovery rates ranged from 81.1-113.7%, and 89.2-115.4%, with the coefficients of variation ranging from 1.4-15.0%, and 1.9-14.8%, respectively. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.97). This work not only developed novel nanocarriers for antibody-based LFIA but also ensured high-performance detection.

A SERS nanocellulose-paper-based analytical device for ultrasensitive detection of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD), one of the most prevalent neurodegenerative diseases, results in severe cognitive decline and irreversible memory loss. Early detection of AD is significant to patients for personalized intervention since effective cure and treatment methods for AD are still lacking. Despite the severity of the disease, existing highly sensitive AD detection methods, including neuroimaging and brain deposit-positive lesion tests, are not suitable for screening purposes due to their high cost and complicated operation. Therefore, these methods are unsuitable for early detection, especially in low-resource settings. Although regular paper-based microfluidics are cost-efficient for AD detection, they are restricted by a poor limit of detection (LOD). RESULTS: To address the above limitations, we report the ultrasensitive and low-cost nanocellulose paper (nanopaper)-based analytical microfluidic devices (NanoPADs) for detecting one of the promising AD blood biomarkers (glial fibrillary acidic protein, GFAP) using Surface-enhanced Raman scattering (SERS) immunoassay. Nanopaper offers advantages as a SERS substrate, such as an ultrasmooth surface, high optical transparency, and tunable chemical properties. We detected the target GFAP in artificial serum, achieving a LOD of 150 fg mL-1. SIGNIFICANCE: The developed NanoPADs are distinguished by their cost-efficiency and ease of implementation, presenting a promising avenue for effective early detection of AD's GFAP biomarker with ultrahigh sensitivity. More importantly, our work provides the experimental routes for SERS-based immunoassay of biomarkers on NanoPADs for various diseases in the future.

Spironolactone metabolite causes falsely increased progesterone in the Abbott Architect immunoassay.

BACKGROUND: Immunoassays are important for routine clinical testing and medical diagnosis. However, they are limited by cross-reactivity especially at low analyte concentrations. There is a critical need to investigate compounds that can interfere with immunoassays. Herein, we describe the identification of canrenone, a spironolactone metabolite that falsely increases progesterone concentrations on the Abbott Architect i2000 Immunoassay. METHODS: Serum samples and assay diluents were spiked with spironolactone or canrenone and progesterone concentrations were measured on the Architect i2000 and Immulite XPi immunoassay platforms. Blood samples from patients taking spironolactone were analyzed with liquid chromatography-tandem mass spectrometry to evaluate the intrinsic response of progesterone concentrations to the presence of canrenone. RESULTS: We measured approximately 10-fold higher progesterone concentrations on the Abbott Architect i2000 compared to reference immunoassay analyzers (Siemens Immulite XPi and Roche Cobas e601/602), suggesting an analytical error which is unique to the Architect i2000 antibody and/or assay conditions. By measuring serum progesterone after addition of spironolactone or canrenone to serum samples, we found that canrenone falsely increased progesterone on the Architect i2000 immunoassay. However, this interference was more pronounced at low serum progesterone concentrations. Moreover, a strong positive correlation was seen between canrenone and measured serum progesterone concentrations. CONCLUSIONS: Our investigations are important for individuals who require progesterone measurements using the Architect i2000 immunoassay, especially because it is unlikely for clinicians to order canrenone measurements alongside progesterone measurements for individuals taking spironolactone. Further research is needed to determine whether canrenone can influence progesterone measurements on other immunoassay systems.

Split-type photoelectrochemical immunoassay for sensitive quantification of carcinoembryonic antigen based on target-induced in situ formation of Z-type heterojunction.

Carcinoembryonic antigen (CEA), a vital biomarker, plays a significant role in the early diagnosis and prognostic estimation of malignant tumors. In this study, a split-type photoelectrochemical immunoassay for the sensitive quantification of CEA has been successfully developed based on the target-induced in situ formation of a Z-type heterojunction. First, gold nanoparticle-decorated ZnIn2S4 (AuNPs/ZnIn2S4) composites were synthesized and used for the fabrication of photoelectrodes. Then, the detection antibody labeled with Ag nanoparticles was formed and applied for the biorecognition of CEA and subsequent liberation of Ag+ ions to induce the in situ formation of Ag2S/AuNPs/ZnIn2S4, a Z-type heterojunction, on the photoelectrode. The Z-type Ag2S/AuNPs/ZnIn2S4 heterojunction with effectively promoted separation of photogenerated charge carriers could lead to a markedly enhanced photocurrent response and highly sensitive quantification of CEA. Moreover, the three-dimensional spatial structure of ZnIn2S4 provides abundant active sites for the reaction and exhibits non-enzymatic properties, which are conducive to the further improvement of the analytical performance of CEA. The developed split-type photoelectrochemical immunoassay with good sensitivity, satisfactory selectivity, reliable stability, wide dynamic linear range (0.01-20 ng mL-1), and low detection limit (7.3 pg mL-1) offers valuable insights into the development of novel PEC biosensing models for the detection of tumor biomarkers and holds potential application value in the field of disease diagnosis.

Prozone masks elevated SARS-CoV-2 antibody level measurements.

We report a prozone effect in measurement of SARS-CoV-2 spike protein antibody levels from an antibody surveillance program. Briefly, the prozone effect occurs in immunoassays when excessively high antibody concentration disrupts the immune complex formation, resulting in a spuriously low reported result. Following participant inquiries, we observed anomalously low measurement of SARS-CoV-2 spike protein antibody levels using the Roche Elecsys® Anti-SARS-CoV-2 S immunoassay from participants in the Texas Coronavirus Antibody Research survey (Texas CARES), an ongoing prospective, longitudinal antibody surveillance program. In July, 2022, samples were collected from ten participants with anomalously low results for serial dilution studies, and a prozone effect was confirmed. From October, 2022 to March, 2023, serial dilution of samples detected 74 additional cases of prozone out of 1,720 participants' samples. Prozone effect may affect clinical management of at-risk populations repeatedly exposed to SARS-CoV-2 spike protein through multiple immunizations or serial infections, making awareness and mitigation of this issue paramount.

Ternary BiOI/Bi2S3/Au Nanosheet Arrays as a Photoelectrochemical Signal Converter for the Detection of Cardiac Troponin I.

Nanosheet arrays with stable signal output have become promising photoactive materials for photoelectrochemical (PEC) immunosensors. However, an essential concern is the facile recombination of carriers in one-component nanoarrays, which cannot be readily prevented, ultimately resulting in weak photocurrent signals. In this study, an immunosensor using gold nanoparticle-anchored BiOI/Bi2S3 nanosheet arrays (BiOI/Bi2S3/Au) as a signal converter was fabricated for sensitive detection of cardiac troponin I (cTnI). The ternary nanosheet arrays were prepared by a simple method in which Bi2S3 was well-coated on the BiOI surface by in situ growth, whereas the addition of Au further improved the photoelectric conversion efficiency and could link more antibodies. The three-dimensional (3D) ordered sheet-like network array structure and BiOI/Bi2S3/Au ternary nanosheet arrays showed stable and high photoelectric signal output and no significant difference in signals across different batches under visible light excitation. The fabricated immunosensor has a sensitive response to the target detection marker cTnI in a wide linear range of 500 fg/mL to 50 ng/mL, and the detection limit was 32 fg/mL, demonstrating good stability and selectivity. This work not only shows the great application potential of ternary heterojunction arrays in the field of PEC immunosensors but also provides a useful exploration for improving the stability of immunosensors.

Electrochemical immunosensing of tumor markers.

The rising incidence and mortality rates of cancer have led to a growing need for precise and prompt early diagnostic approaches to effectively combat this disease. However, traditional methods employed for detecting tumor cells, such as histopathological and immunological techniques, are often associated with complex procedures, high analytical expenses, elevated false positive rates, and a dependence on experienced personnel. Tracking tumor markers is recognized as one of the most effective approaches for early detection and prognosis of cancer. While onco-biomarkers can also be produced in normal circumstances, their concentration is significantly elevated when tumors are present. By monitoring the levels of these markers, healthcare professionals can obtain valuable insights into the presence, progression, and response to treatment of cancer, aiding in timely diagnosis and effective management. This review aims to provide researchers with a comprehensive overview of the recent advancements in tumor markers using electrochemical immunosensors. By highlighting the latest developments in this field, researchers can gain a general understanding of the progress made in the utilization of electrochemical immunosensors for detecting tumor markers. Furthermore, this review also discusses the current limitations associated with electrochemical immunosensors and offers insights into paving the way for further improvements and advancements in this area of research.

Polylevodopa nanoplatform for lateral flow immunochromatography assay of SARS-CoV-2 and influenza A virus.

At the end of 2019, an unprecedented outbreak of novel coronavirus pneumonia ravaged the global landscape, inflicting profound harm upon society. Following numerous cycles of transmission, we find ourselves in an epoch where the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coexists alongside influenza viruses (Flu A). Swift and accurate diagnosis of SARS-CoV-2 and Flu A is imperative to stem the spread of these maladies and administer appropriate treatment. Presently, colloidal gold-based lateral flow immunoassays (Au-LFIAs) constructed through electrostatic adsorption are beset by challenges such as diminished sensitivity and feeble binding stability. In this context, we propose the adoption of black polylevodopa nanoparticles (PLDA NPs) featuring abundant carboxyl groups as labeling nanomaterials in LFIA to bolster the stability and sensitivity of SARS-CoV-2 antigens and influenza A virus identifications. The engineered PLDA-LFIAs exhibit the capacity to detect SARS-CoV-2 and Flu A within 30 min, boasting a detection threshold of 5 pg/ml for the SARS-CoV-2 antigen and 0.1 ng/ml for the Flu A H1N1 antigen, thereby underscoring their heightened sensitivity relative to Au-LFIAs. These PLDA-LFIAs hold promise for the early detection of SARS-CoV-2 and Flu A, underscoring the potential of PLDA NPs as a discerning labeling probe to heighten the sensitivity of LFIA across diverse applications.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 µL and 100 µL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

Click Reaction-Mediated Fluorescent Immunosensor Based on Cu-MOF Nanoparticles for Ultrasensitive and High-Throughput Detection of Aflatoxin B1 in Food Samples.

Due to the high toxicity of aflatoxin B1 and its risks to human health, we developed a click reaction-mediated automated fluorescent immunosensor (CAFI) for sensitive detection of aflatoxin B1 based on the Cu(I)-catalyzed click reaction. With its large specific surface area, a copper-based metal-organic framework (Cu-MOF) was synthesized to adsorb and enrich the copper ion (Cu(II)) and then load the complete antigen (BSA-AFB1). After the immunoreaction, Cu(II) inside the Cu-MOF-Antigen conjugate would be reduced to Cu(I) in the presence of sodium ascorbate, which triggered the click reaction between the fluorescent donor-modified DNA and the receptor-modified complementary DNA to lead to a fluorescence signal readout. The whole reaction steps were finished by the self-developed automated immunoreaction device. This CAFI method showed a limit of detection (LOD) of 0.48 pg/mL as well as a 670-fold enhancement in sensitivity compared to conventional ELISA, revealing its great potential in practical applications and automated detection.

Comparison of single and double pulse laser-induced breakdown spectroscopy for the detection of biomolecules tagged with photon-upconversion nanoparticles.

BACKGROUND: Laser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. RESULTS: This paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL-1 to 0.29 ng mL-1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. SIGNIFICANCE AND NOVELTY: In this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.