Traditional Enzyme-linked Immunosorbent Assay Research Articles

A dual-aptamer sandwich assay by detection of c-reactive protein in synovial fluid on an integrated microfluidic system for diagnosis of periprosthetic joint infection

Diagnosing periprosthetic joint infection (PJI) in equivocal cases is always challenging. Synovial fluid (SF) C-reactive protein (CRP) levels have been reported as a surrogate to improve the diagnosis accuracy. This study reported a new integrated microfluidic system (IMS) designed to detect SF-CRP at a dynamic range of 1-50mg/L. The novel, magnetic bead-based, dual-aptamer assay with two CRP-specific aptamers, St1 and St2, enabled rapid and accurate CRP detection. This represents the first attempt to automatically perform the CRP dual-aptamer assay on a microfluidic chip quantifying CRP in SF with optical signals. CRP concentrations in fourteen clinical samples were accurately quantified by using the dual-aptamer assay on the developed IMS in an automatic format, suggesting the assay’s potential as a reliable diagnostic tool for SF-CRP detection and may serve as an armamentarium for PJI diagnosis. Notably, the assay was deemed superior to traditional enzyme-linked immunosorbent assay (ELISA) because it consumes a smaller amount of sample (only 40μL), requires fewer reagents (only 50μL of secondary detection aptamer and horseradish peroxidase), and provides quicker results (~2hours).

A Multiplexed Digital Platform Enables Detection of Attomolar Protein Levels with Minimal Cross-Reactivity.

Protein-based biomarkers are essential for disease diagnostics, yet their low abundance in biofluids often presents significant detection challenges for traditional enzyme-linked immunosorbent assay (ELISA) techniques. While various ultrasensitive methods such as digital ELISA have improved sensitivity, multiplex assays still suffer from considerable cross-reactivities that can compromise result accuracies. To address this challenge, we have developed barcoded Molecular On-bead Signal Amplification for Individual Counting (barcoded MOSAIC), a multiplexed digital ELISA technology that markedly reduces cross-reactivity by pairing barcoded detection antibodies with specific bead types. This approach enables the simultaneous detection of eight analytes from less than 9 μL of blood, with sensitivities ranging from midpicomolar to low-attomolar levels and a collective dynamic range exceeding seven logs across multiple analytes within a single multiplex assay. Additionally, barcoded MOSAIC is compatible with standard immunoassay reagents and workflows, utilizing a rapid, automatable flow cytometric readout for quantification, which makes it a highly accessible benchtop platform that is readily adoptable by both research and clinical laboratories, setting the stage for future translation into point-of-care applications.

Development of a fully automated chemiluminescent immunoassay for the quantitative and qualitative detection of antibodies against African swine fever virus p72.

African swine fever (ASF), caused by ASF virus (ASFV), is a highly infectious and severe hemorrhagic disease of pigs that causes major economic losses. Currently, no commercial vaccine is available and prevention and control of ASF relies mainly on early diagnosis. Here, a novel automated double antigen sandwich chemiluminescent immunoassay (DAgS-aCLIA) was developed to detect antibodies against ASFV p72 (p72-Ab). For this purpose, recombinant p72 trimer was produced, coupled to magnetic particles as carriers and labeled with acridinium ester as a signal trace. Finally, p72-Ab can be sensitively and rapidly measured on an automated chemiluminescent instrument. For quantitative analysis, a calibration curve was established with a laudable linearity range of 0.21 to 212.0 ng/mL (R2 = 0.9910) and a lower detection limit of 0.15 ng/mL. For qualitative analysis, a cut-off value was set at 1.50 ng/mL with a diagnostic sensitivity of 100.00% and specificity of 98.33%. Furthermore, antibody response to an ASF gene-deleted vaccine candidate can be accurately quantified using this DAgS-aCLIA, as evidenced by early seroconversion as early as 7 days post-immunization and high antibody levels. Compared with available enzyme-linked immunosorbent assays, this DAgS-aCLIA demonstrated a wider linearity range of 4 to 16-fold, and excellent analytical sensitivity and agreement of over 95.60%. In conclusion, our proposed DAgS-aCLIA would be an effective tool to support ASF epidemiological surveillance.IMPORTANCEAfrican swine fever virus (ASFV) is highly contagious in wild boar and domestic pigs. There is currently no vaccine available for ASF, so serological testing is an important diagnostic tool. Traditional enzyme-linked immunosorbent assays provide only qualitative results and are time and resource consuming. This study will develop an automated chemiluminescent immunoassay (CLIA) that can quantitatively and qualitatively detect antibodies to ASFV p72, greatly reducing detection time and labour-intensive operation, and improving detection sensitivity and linearity range. This novel CLIA would serve as a reliable and convenient tool for ASF pandemic surveillance and vaccine development.

A-351 Versatile and Sensitive Microfluidic Immunoassay for Improved Point-of-Care Diagnostics

Abstract Background Healthcare professionals must choose between sensitivity and ease-of-use in diagnostics, particularly for diseases like COVID-19 and strep throat. Traditional enzyme-linked immunosorbent assays (ELISAs) are sensitive but are manually intensive and lengthy. Lateral flow assays (LFAs) are an alternative with low cost, ease-of-use, and rapid results; but LFAs suffer from low sensitivity. Burst Diagnostics presents a groundbreaking solution with the capillary-driven immunoassay (CaDI), a novel microfluidic device delivering sensitivity comparable to ELISA with the ease of LFAs. Methods The CaDI is made of flow channels defined by layered transparency and adhesive sheets containing reagent pads with HRP-conjugated antibody and luminol. The system requires only a sample addition step by the user. Sample flow initiates sequential steps of a sandwich ELISA, fully automated by the device, to provide a chemiluminescent signal in 10 minutes or less, depending on sample matrix. Results We have demonstrated success with preliminary studies for the detection of SARS-CoV-2 nucleocapsid protein (NP) and group A streptococcus (GAS). Detection of NP was achieved by assembling and optimizing COVID-19 CaDI’s using anti-NP antibodies. Then, spiked nasal swab samples were tested ranging from 10 to 1000 PFU/mL with positive detection as low as 10 PFU/mL, much less than the same samples in a traditional LFA (see figure). Additionally, we have demonstrated successful detection of GAS in optimized buffer conditions up to 100-fold less than commonly used LFAs. Finally, multiplexed CaDI’s increase throughput and cost-effectiveness in the point-of-care setting. Conclusions The CaDI platform bridges the ease of LFAs and sensitivity/specificity of laboratory tests. Currently, the CaDI can detect SARS-CoV-2 NP and GAS with higher sensitivity than existing LFAs. Development is underway for a reader to accompany the CaDI and further simplify the test. Importantly, the potential targets for detection are immense due to the sensitivity, simplicity, and adaptability of the platform.

ELISA-R: an R-based method for robust ELISA data analysis.

Enzyme-linked immunosorbent assay (ELISA) is a technique to detect the presence of an antigen or antibody in a sample. ELISA is a simple and cost-effective method that has been used for evaluating vaccine efficacy by detecting the presence of antibodies against viral/bacterial antigens and diagnosis of disease stages. Traditional ELISA data analysis utilizes a standard curve of known analyte, and the concentration of the unknown sample is determined by comparing its observed optical density against the standard curve. However, in the case of vaccine research for complicated bacteria such as Mycobacterium tuberculosis (Mtb), there is no prior information regarding the antigen against which high-affinity antibodies are generated and therefore plotting a standard curve is not feasible. Consequently, the analysis of ELISA data in this instance is based on a comparison between vaccinated and unvaccinated groups. However, to the best of our knowledge, no robust data analysis method exists for "non-standard curve" ELISA. In this paper, we provide a straightforward R-based ELISA data analysis method with open access that incorporates end-point titer determination and curve-fitting models. Our modified method allows for direct measurement data input from the instrument, cleaning and arranging the dataset in the required format, and preparing the final report with calculations while leaving the raw data file unchanged. As an illustration of our method, we provide an example from our published data in which we successfully used our method to compare anti-Mtb antibodies in vaccinated vs non-vaccinated mice.

Development of a multiplexing method for the quantification of “high-risk” host cell lipases in biotherapeutics by Luminex

Clearance of residual Host Cell Proteins (HCPs) is critical for the manufacturing processes of biotherapeutics. HCPs have the potential to impact product efficacy and quality, posing a risk to patient safety. It is therefore essential to be able to both identify and quantitate HCPs throughout drug development, even if the proteins are present in low concentrations. Traditional Enzyme-Linked Immunosorbent Assays (ELISAs) have historically served as the gold standard for monitoring HCPs; however, ELISA methods are labor-intensive and costly. With an increase of HCPs being identified below detectable quantification levels, there is a need for simultaneous detection of selectively targeted HCPs. Here, we develop a Luminex multiplexing method that is able to accurately quantify two “high-risk” lipases Lipoprotein Lipase (LPL) and Phospholipase B-Like 2 (PLBL2) within the same assay. This study outlines the method development for optimizing parameters such as antibody constructs, conjugation ratios, signal enhancement, and more in order to create the most efficient multiplexing method. As a result, a Luminex multiplexing method can provide a similar result to a monoplexing ELISA method but in a faster and more cost-effective manner. This method can be expanded to include other “high-risk” HCPs and used for future HCP applications.

Dual improved enzyme-linked immunosorbent assay based on multivalent bifunctional nanobody and polymerized horseradish peroxide for the ultrasensitive detection of Ochratoxin A in coffee samples

Enzyme-linked immunosorbent assay (ELISA) is favored for its high throughput, low cost, and simplicity, but the limited sensitivity of the traditional colorimetric ELISA restricts its wider application. Therefore, developing improvement strategies to enhance its sensitivity is a current research hotspot. In this work, we developed a multivalent bifunctional nanobody (Nb28-C4-SBP) by fusing a nanobody gene (Nb28) against ochratoxin A (OTA) with a self-assembling peptide (C4) and a streptavidin-conjugated peptide (SBP), which can introduce streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) as a probe into the detection system. Subsequently, a dual improved ELISA (DI-ELISA) was developed using the higher affinity multivalent bifunctional nanobody (Nb28-C4-SBP) and the more enzyme-loaded probe (SA-PolyHRP) for the ultrasensitive detection of OTA in coffee samples. The DI-ELISA demonstrated a detection limit of 1 pg/mL, which was about 20-fold lower than that of the Nb28-C4-SBP and SA-HRP based ELISA and 266-fold lower than that of the Nb28-SBP and SA-PolyHRP based ELISA, respectively. In summary, we developed an ultrasensitive, rapid, and simple immunoassay for detecting OTA in coffee samples. Additionally, the proposed improvement strategy based on multivalent nanobody and polymerized horseradish peroxide is straightforward to operate and holds great potential for enhancing the sensitivity in various immunoassays.

Development of two novel ELISAs based on Prussian blue nanoparticles for ultrasensitive detection of norfloxacin in milk

The development of traditional enzyme-linked immunosorbent assay (ELISA) systems for the detection of small-molecule residues in foods is limited because of the poor stability of biological enzymes and the lower sensitivity of absorption-based signals. Herein, two ELISAs based on Prussian blue nanoparticles (PBNPs) were developed to establish ultrasensitive and stable methods for detecting norfloxacin (NOR) in milk. The results show that the detection limit (IC15) of NOR was 0.77 μg L−1 and the sensitivity (IC50) was 18.28 μg L−1 in the standard solution using the PBNPs-based nano-ELISA. When the fluorescence quenching ELISA based on PBNPs was used, the detection limit was 0.06 μg L−1 and the sensitivity was 4.21 μg L−1 in the standard solution. The recoveries and precision were good, as confirmed by analysis of real milk samples. The results were consistent with those of commercial ELISA kits, indicating the high accuracy of these two methods.

3D Film-Like Nanozyme with a Synergistic Amplification Effect for the Ultrasensitive Immunochromatographic Detection of Respiratory Viruses.

Greatly improving the sensitivity and detection range of lateral flow immunoassays (LFAs) by at least 100 times without using additional instruments remains challenging. Herein, we develop a three-dimensional (3D) film-like nanozyme (GO-Pt30-AuPt5) by ordered assembly of one layer of 30 nm Pt nanoparticles (NPs) and one layer of small Au@Pt satellites (5 nm) onto a two-dimensional (2D) graphene oxide (GO) nanofilm, in which GO greatly increased the interface area and stability of the nanozyme whereas Pt and Au@Pt NPs synergistically enhanced colorimetric/catalytic activities. The grafting of outer Au@Pt satellites converted the 2D nanofilm into a 3D flexible nanozyme with numerous catalytic sites for enzymatic deposition signal amplification and binding sites for target capture. The introduction of GO-Pt30-AuPt5 into multiplex LFA achieved the ultrasensitive and simultaneous detection of two important respiratory viruses with sensitivity of 1 pg/mL level, which was about 100 times higher than that without signal enrichment and at least 20 and 1900 times higher than those of traditional enzyme-linked immunosorbent assay and AuNP-based LFA, respectively. The clinical utility of the proposed assay was validated through the diagnosis of 49 real clinical respiratory tract specimens. Our proposed LFA shows great potential for the ultrasensitive screening of pathogens in the field.

In situ growth of double-hybrid nanoflowers on paper for the visual detection of carcinoembryonic antigen

In this study, the hemoglobin, egg white, copper phosphate organic–inorganic hybrid nanoflowers (H-E-Cu-NFs), and filter paper-based-egg white-Ca3(PO4)2 HNFs (FP-E-Ca-NFs) were synthesized using a mild biomineralization method. Antibodies (Ab) were labeled on the surface of nanoflowers through highly specific interactions between avidin and biotin. The FP-E-Ca-NFs@Biotin N-hydroxysuccinimide ester–Anti-carcinoembryonic antigen primary antibodies (Biotin-NHS-Ab1)/carcinoembryonic antigen (CEA)/H-E-Cu-NFs@Biotin-NHS-Ab2 immune-biosensing system was constructed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to achieve the visual detection of CEA. Among them, H-E-Cu-NFs have abundant enzyme-like catalytic sites of Fe3+/Cu2+, which can effectively improve the efficiency of the colorimetric reaction. In contrast, E-Ca-NFs have no catalytic sites, and the directional design of catalytic performance realizes the specific identification and signal amplification of CEA detection. The as-developed immune-biosensing system showed a detection range of 0.5 ∼ 100 ng/mL, with the lowest detection limit of 0.5 ng/mL and the CEA recovery between 89.5 % ∼ 108.1 % in human serum samples. Compared to traditional ELISA methods, this immune-biosensing system has the advantages of easy operation, low cost, rapid detection, and on-site detection.

Revolutionizing ELISA: A one-step blocking approach using lipid bilayer coatings

Enzyme-linked immunosorbent assay (ELISA) is an essential technique for biomolecule detection in diagnostics and research, but traditional protocols are time-consuming and variable. Conventional blocking agents like bovine serum albumin (BSA) pose ethical issues and potential assay interference. This study presents a one-step lipid-blocking approach to simplify the ELISA procedure. Compared to conventional blockers, the Lipid Universal Coating Assembly (LUCA) antifouling blocker showed comparable sensitivity while significantly reducing processing time and steps. Furthermore, it showed improved co-blocking efficacy and signal intensity in conventional protocols when combined with minimal BSA concentrations. Stability tests under accelerated aging confirmed the robustness of the LUCA antifouling blocker. Additionally, it effectively facilitated antibody detection for COVID-19 antigens in direct ELISA and human IL-6 antigen in Sandwich ELISA, suggesting its versatile applicability. Taken together, our findings reveal that one-step lipid blocking not only streamlines the ELISA process but also maintains high sensitivity and specificity, providing an efficient, user-friendly, and environmentally friendly alternative to traditional ELISA blockers and a robust platform for rapid diagnostics.

Toward the point-of-care testing of alkaline phosphatase in human serum

This work addresses challenges of simple but reliable determination of enzyme alkaline phosphatase (ALP) in μL-sized serum samples. The single-tube assay and plate-based immunoassay were developed by using a commercial glucose test strip (GTS) to transduce analytical signal from the ALP + glucose-6-phosphate (G6P) reaction. The glucose released from G6P contributed to the flow of anodic charge Q20s through GTS. The interferences from serum matrix including those from native glucose were eliminated by devising a signal ΔQ20s = Q20s (total) – Q20s (matrix). The ΔQ20s was proportional to ALP activity within a linear range of 7.4–720 IU L−1 for assay (R2, 0.995) and 10–300 IU L−1 for immunoassay (R2, 0.983), which far exceeded a normal clinical range for ALP in adult human serum (30–150 IU L−1). Both methods required no auxiliary enzymes or labels, and were accurate (93–99 % signal recovery) and precise (RSD ≤10%). The assay was a simpler option because it required only one 20-min incubation step to determine ALP. The immunoassay involved three steps but it was still less laborious (by ∼70%) than a traditional enzyme-linked immunosorbent assay (ELISA) of ALP due to the substitution of ELISA detection antibody with GTS. The GTS-G6P-based methods bring ALP testing closer to the point-of-care locations, which is important considering the growing role of ALP as a prophylactic, therapeutic, and anti-inflammatory agent, and a marker of human diseases.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Diagnostic Accuracy of IgA Anti-Transglutaminase Assessed by Chemiluminescence: A Systematic Review and Meta-Analysis.

A new chemiluminescence immunoassay method (CLIA) for detecting IgA anti-transglutaminase (atTG IgA) in celiac disease (CD) has prompted inquiries into its diagnostic performance. We conducted a systematic review and meta-analysis comparing CLIA with traditional enzyme-linked immunosorbent assay (ELISA) and fluorescence enzyme immunoassay (FEIA). We searched PubMed, Medline, and Embase databases up to March 2024. The diagnostic references were intestinal biopsy and ESPGHAN guidelines. We calculated the sensitivity and specificity of atTG IgA assessed by CLIA and the odds ratio (OR) between the assays. Eleven articles were eligible for the systematic review and seven for the meta-analysis. Sensitivity and specificity of atTG IgA CLIA-assay were 0.98 (95% CI, 0.95-0.99) and 0.97 (95% CI, 0.94-0.99), respectively. The sensitivity of atTG IgA antibody detection did not significantly vary across the three assay modalities examined (CLIA vs. ELISA OR: 1.08 (95% CI, 0.56-2.11; p = 0.8); CLIA vs. FEIA OR: 6.97 (95% CI, 0.60-81.03; p = 0.1). The specificity of atTG IgA assessed by FEIA was higher than for CLIA (OR 0.17 (95% CI, 0.05-0.62); p < 0.007). According to the systematic review, normalization of atTG IgA levels in CD patients following a gluten-free diet was delayed when using CLIA compared to ELISA and FEIA methods. Conflicting findings were reported on the antibody threshold to use in order to avoid biopsy confirmation.

Label-Free and Ultrasensitive Detection of Cartilage Acidic Protein 1 in Osteoarthritis Using a Single-Walled Carbon Nanotube Field-Effect Transistor Biosensor.

Osteoarthritis (OA), a prevalent degenerative joint disease, significantly affects the well-being of afflicted individuals and compromises the standard functionality of human joints. The emerging biomarker, Cartilage acidic protein 1 (CRTAC1), intricately associates with OA initiation and serves as a prognostic indicator for the trajectory toward joint replacement. However, existing diagnostic methods for CRTAC1 are hampered by the limited abundance, thus restricting the precision and specificity. Herein, a novel approach utilizing a single-walled carbon nanotube field-effect transistor (SWCNTs FET) biosensor is reported for the direct label-free detection of CRTAC1. High-purity semiconducting carbon nanotube films, functionalized with antibodies of CRTAC1, provide excellent electrical and sensing properties. The SWCNTs FET biosensor exhibits high sensitivity, notable reproducibility, and a wide linear detection range (1 fg/mL to 100 ng/mL) for CRTAC1 with a theoretical limit of detection (LOD) of 0.2 fg/mL. Moreover, the SWCNTs FET biosensor is capable of directly detecting human serum samples, showing excellent sensing performance in differentiating clinical samples from OA patients and healthy populations. Comparative analysis with traditional enzyme-linked immunosorbent assay (ELISA) reveals that the proposed biosensor demonstrates faster detection speeds, higher sensitivity/accuracy, and lower errors, indicating high potential for the early OA diagnosis. Furthermore, the SWCNTs FET biosensor has good scalability for the combined diagnosis and measurement of multiple disease markers, thereby significantly expanding the application of SWCNTs FETs in biosensing and clinical diagnostics.

Modulating Electron Transfer between Pt and MOF Support through Pd Doping Promotes Nanozyme Catalytic Efficiency.

Electron transfer is considered to be a typical parameter that affects the catalytic activity of nanozymes. However, there is still controversy regarding whether higher or lower electron transfer numbers are beneficial for improving the catalytic activity of nanozymes. To address this issue, we propose the introduction of Pd doping as an important electron regulation strategy to tune electron transfer between Pt and ZIF-8 carriers (PtxPd1@ZIF-8). We observe a volcano-shaped relationship between the electron transfer number and catalytic activity, reaching its peak at Pt4Pd1@ZIF-8. Mechanism studies indicate that as the electron transfer number from Pt to ZIF-8 carriers increases, the d-band center of the active site Pt increases, reducing the occupancy of antibonding states and enhancing the adsorption capacity of the key intermediate (*O). However, a further increase in the adsorption of *O energy makes it difficult to desorb and participate in the next reaction, thus exhibiting volcanic activity. The optimized Pt4Pd1@ZIF-8 nanozyme is applied to develop an immunoassay for the detection of zearalenone, achieving a detection limit of 0.01 μg/L, which is 6 times higher than that of the traditional enzyme-linked immunosorbent assay. This work not only reveals the potential regulatory mechanism of electron transfer on the catalytic activity of nanozymes but also improves the performance of nanozyme-based biosensors.

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.

Rapid Homogeneous Fluorescent Enzyme-Linked Immunosorbent Assay (ELISA) for N-Terminal Brain Natriuretic Peptide (NT-proBNP) Combining Magnetic Probes and Ampliflu Red

Heart failure (HF) is the leading cause of death from cardiovascular disease, and early diagnosis using serum markers holds extraordinary significance for the treatment of patients. N-terminal brain natriuretic peptide (NT-proBNP) is the gold standard for HF diagnosis. Herein, a novel, rapid, sensitive, and immune-sensing strategy for NT-proBNP is reported employing an enzyme-linked immunosorbent assay (ELISA) that combines the use of magnetic biological probes and the fluorogenic substrate Ampliflu Red. Functionalized immunomagnetic nanoparticles with large surface areas provide an abundant reaction platform and reduce the analysis time due to their superparamagnetic properties. Ampliflu Red is catalyzed by horseradish peroxidase (HRP) using hydrogen peroxide (H2O2) to produce strong fluorescence and a low background. The reported method achieved a good detection limit of 2.73 ng mL−1 and a wide linear range from 3.13 to 100 ng mL−1. The analysis time was less than 1 h, which is superior to that of traditional ELISA (3–4 h). In addition, the recoveries of spiked human serum were satisfactory, indicating that the proposed strategy has potential applications for the rapid determination of NT-proBNP and other biomarkers in clinical settings.

A point-of-care electrochemical biosensor for the rapid and sensitive detection of biomarkers in murine models with LPS-induced sepsis

Sepsis is a life-threatening condition, which is irreversible if diagnosis and intervention are delayed. The response of the immune cells towards an infection triggers widespread inflammation through the production of cytokines, which may result in multiple organ dysfunction and eventual death. Conventional detection techniques fail to provide a rapid diagnosis because of their limited sensitivity and tedious protocol. This study proposes a point-of-care (POC) electrochemical biosensor that overcomes the limitations of current biosensing technologies in the clinical setting by its integration with electrokinetics, enhancing the sensitivity to picogram level compared with the nanogram limit of current diagnostic technologies. This biosensor promotes the use of a microelectrode strip to address the limitations of conventional photolithographic fabrication methods. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and microRNA-155 (miR-155) were monitored in a lipopolysaccharide (LPS)-induced septic mouse model. The optimum target hybridization time in a high conductivity medium was observed to be 60 s leading to the completion of the whole operation within 5 min compared with the 4-h detection time of the traditional enzyme-linked immunosorbent assay (ELISA). The limit of detection (LOD) was calculated to be 0.84, 0.18, and 0.0014 pg mL−1, respectively. This novel sensor may have potential for the early diagnosis of sepsis in the clinical setting.

Advanced detection of cervical cancer biomarkers using engineered filamentous phage nanofibers.

Cervical cancer is a major global health concern, characterized by its high incidence and mortality rates. The detection of tumor markers is crucial for managing cancer, making treatment decisions, and monitoring disease progression. Vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PDL-1) are key targets in cervical cancer therapy and valuable biomarkers in predicting treatment response and prognosis. In this study, we found that combining the measurement of VEGF and soluble PDL-1 can be used for diagnosing and evaluating the progression of cervical cancer. To explore a more convenient approach for detecting and assessing cervical cancer, we designed and prepared an engineered fd bacteriophage, a human-safe viral nanofiber, equipped with two peptides targeting VEGF and PD-L1. The dual-display phage nanofiber specifically recognizes and binds to both proteins. Utilizing this nanofiber as a novel capture agent, we developed a new enzyme-linked immunosorbent assay (ELISA) method. This method shows significantly enhanced detection sensitivity compared to conventional ELISA methods, which use either anti-VEGF or anti-PD-L1 antibodies as capture agents. Therefore, the phage dual-display nanofiber presents significant potential in detecting cancer markers, evaluating medication efficacy, and advancing immunotherapy drug development. KEY POINTS: • The combined measurement of VEGF and soluble Programmed Death-Ligand 1(sPD-L1) demonstrates an additive effect in the diagnosis of cervical cancer. Fd phage nanofibers have been ingeniously engineered to display peptides that bind to VEGF and PD-L1, enabling the simultaneous detection of both proteins within a single assay • Genetically engineered phage nanofibers, adorned with two distinct peptides, can be utilized for the diagnosis and prognosis of cancer and can be mass-produced cost-effectively through bacterial infections • Employing dual-display fd phage nanofibers as capture probes, the phage ELISA method exhibited significantly enhanced detection sensitivity compared to traditional sandwich ELISA. Furthermore, phage ELISA facilitates the detection of a single protein or the simultaneous detection of multiple proteins, rendering them powerful tools for protein analysis and diagnosis across various fields, including cancer research.