Multiplex Biomarker Research Articles

Plasma proteome profiling in giant cell arteritis

ObjectivesThis study aimed to identify plasma proteomic signatures that differentiate active and inactive giant cell arteritis (GCA) from non-disease controls. By comprehensively profiling the plasma proteome of both patients with...

Abstract A072: Oncuria®-Monitor, a non-invasive test for the detection of recurrent bladder cancer: Cross sectional performance evaluation from a multicenter study

Abstract Up to 70% of patients with non-muscle invasive bladder cancer (NMIBC) experience disease recurrence, making it one of the most prevalent cancers in the US. The purpose of this study was to test the performance of Oncuria®-Monitor, a multiplex urinary biomarker assay for the monitoring of voided urine for recurrent bladder cancer. This cross-sectional, multicenter study included a total of 283 subjects with a history of bladder cancer. Voided urine specimens were collected prior to any procedures for multiplex analysis. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values (e.g., sensitivity and specificity) and compared with the patients’ clinical data and final diagnosis as defined by the results of cystoscopy. Oncuria®-Monitor achieved an overall sensitivity of 94.3%, specificity of 83.6% and negative predictive valuer (NPV) of 96.2% for the detection of recurrent bladder cancer. Patient summary: We conducted a large cross-sectional clinical study for detecting recurrent bladder cancer with a novel, simple urine test (Oncuria®- Monitor), which measures 10 bladder cancer associated proteins in a single voided urine and can be used to detect recurrent bladder cancer. We analyzed urines from 283 patients and demonstrated that Oncuria®-Monitor can detect recurrent bladder cancer with a high degree of accuracy, specifically in patients with high grade or high stage disease. In conclusion, the novel Oncuria®-Monitor urine test can be used to surveil patients with a history of bladder cancer. Citation Format: Zhen Zhang, Florence Le Calvez-Kelm, Makito Miyake, Ian Pagano, Takuto Shimizu, Sergei Tikhonenkov, Sunao Tanaka, Charles J Rosser, Steve Goodison, Hideki Furuya. Oncuria®-Monitor, a non-invasive test for the detection of recurrent bladder cancer: Cross sectional performance evaluation from a multicenter study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A072.

Early-stage Alzheimer’s disease profiling in blood achieved by multiplexing aptamer-SERS biosensors

Neurological disorders are the second leading cause of death globally, with Alzheimer’s disease (AD) emerging as a significant contributor, responsible for 276 million cases in disability-adjusted life years. Conventional diagnostic methods are often invasive, costly, and place a considerable strain on global healthcare systems. In this study, we presented an innovative and efficient strategy for AD assessment through blood profiling using a multiwell glass chip integrated with aptamer-based surface-enhanced Raman scattering (SERS) biosensors. High-affinity aptamers were selected using capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX). A mouse brain injury model was employed to systematically investigate biomarkers indicative of physiological, vascular, and cellular damage, such as neurogranin (Nrgn), angiopoietin-2 (Angio-2), PRDX3, lactate dehydrogenase (L-LDH), and τ-441, which were quantified at atto-molar levels in blood samples. Additionally, with the aid of CT-scan imaging, an aptamer-SERS assay was developed to evaluate the dynamic regulation of AD biomarkers. The aptamer-SERS biosensor system was also applied to human samples, demonstrating its capability to multiplex AD biomarkers and establish a time-dependent correlation between percentage biomarker regulation and disease progression. The innovative design, fabrication of aptamer-SERS nanoprobes, and the bio-sensing outcomes illustrate the strong potential of this approach for selective, sensitive, and quantitative early-stage AD diagnosis in clinical applications.

Comprehensive serum biomarker analysis reveals IL-8 changes as the only predictor of the effectiveness of immune checkpoint inhibitors for patients with advanced non-small cell lung cancer

ObjectivesProgrammed cell death ligand 1 (PD-L1) expression is widely used to predict the effectiveness of PD-(L)1 inhibitors despite its imperfection. Previous studies suggested the utilization of various serum biomarkers; nonetheless, findings are inconclusive because of limited sample sizes or the focus on a single biomarker in many of these studies. This study analyzed multiplex serum biomarkers to explore their predictive ability in a large cohort of patients with advanced non-small-cell lung cancer (NSCLC) treated with a PD-L1 inhibitor in a real-world setting. Materials and MethodsThis was a sub-study of J-TAIL, a prospective observational study of atezolizumab monotherapy in pre-treated patients with advanced NSCLC. From April to October 2019, 262 patients were enrolled from 73 sites in Japan. Serum samples were collected at baseline and at the second dose of atezolizumab. Quantification of the 51 serum cytokines, chemokines, growth factors, and vascular endothelial growth factors was performed using the Luminex platform. Baseline values and fold changes of the time of the second dose to the baseline were examined in association with the effectiveness of atezolizumab. ResultsAmong the 51 proteins assessed, a higher baseline interleukin (IL)-12 level, a higher soluble CD40 ligand fold change, a lower IL-8 fold change were associated with higher objective response rate (ORR). Of these, only the lower IL-8 fold change was associated with better progression-free survival (PFS) (adjusted hazard ratio, 1.98; 95 % confidence interval, 1.45–2.70; P < 0.01). Multivariate analysis demonstrated that the lower IL-8 fold change was an independent factor for both the ORR and PFS. The IL-8 fold change was independent of the neutrophil/lymphocyte ratio, and durable PFS was observed in patients with both low. ConclusionComprehensive serum biomarker analysis revealed that a lower fold change in serum IL-8 was associated with better outcomes in pre-treated patients with advanced NSCLC receiving atezolizumab.

Portable, Facile Microfluidic Platform Using a Low-Cost Vacuum Pump for Multiplex Biomarker Detection in Human Samples

Portable multiplex microfluidic platforms are critically needed for point-of-care testing (POCT) of health-related biomarkers. The complex preparation processes and the use of expensive and bulky devices lead to increased detection costs, hindering the application of emerging POCT technologies. Here, we introduce a portable and versatile microfluidic platform for low-cost multiplex biomarker detection, integrated with a facile and easy-to-operated vacuum pump and mass-produced screen-printed carbon electrodes (SPCEs). The innovative pump, comprising only a syringe, a valve, and a spring, costs approximately $0.50. It operates by using the compressed spring to self-sufficiently drive reagents, which are actuated via a valve switch. The pump's performance was assessed under various conditions, demonstrating consistent operation and a broad pressure range. Additionally, electrocatalytic nanocomposites (Ti3C2Tx/Pt-Pd) were adopted to modify the SPCEs, resulting in high-efficiency performance (<15min), cost-effectiveness (<$4 per test) and excellent reliability across a broad detection scope. Finally, the platform successfully achieved multiplex detection of biomarkers (glucose, uric acid and lactic acid) in serum, sweat and saliva. This simple and adaptable platform promises to facilitate cost-effective, multiplex biomarker detection, paving the way for large-scale POCT applications, particularly in resource-limited settings.

Transdermal Minimally Invasive Optical Multiplex Detection of Protein Biomarkers by Nanopillars Array-Embedded Microneedles.

Biomarkers detection has become essential in medical diagnostics and early detection of life-threatening diseases. Modern-day medicine relies heavily on painful and invasive tests, with the extraction of large volumes of venous blood being the most common tool of biomarker detection. These tests are time-consuming, complex, expensive and require multiple sample manipulations and trained staff. The application of "intradermal" biosensors utilizing microneedles as minimally invasive sensing elements for capillary blood biomarkers detection has gained extensive interest in the past few years as a central point-of-care (POC) detection platform. Herein, we present a diagnosis paradigm based on vertically aligned nanopillar array-embedded microneedles sampling-and-detection elements for the direct optical detection and quantification of biomarkers in capillary blood. We present here a demonstration of the simple fabrication route for the creation of a multidetection-zone silicon nanopillar array, embedded in microneedle elements, followed by their area-selective chemical modification, toward the multiplex intradermal biomarkers detection. The utilization of the rapid and specific antibody-antigen binding, combined with the intrinsically large sensing area created by the nanopillar array, enables the simultaneous efficient ultrafast and highly sensitive intradermal capillary blood sampling and detection of protein biomarkers of clinical relevance, without requiring the extraction of blood samples for the ex vivo biomarkers analysis. Through preliminary in vitro and in vivo experiments, the direct intradermal in-skin blood extraction-free platform has demonstrated excellent sensitivity (low pM) and specificity for the accurate multiplex detection of protein biomarkers in capillary blood.

Multiplex Collagen Fingerprinting for the Staging of Hepatic Fibrosis Using High-Precision Fluorescence-Guided SERS Imaging.

Hepatic fibrosis is a common chronic liver disease, and its severe progression can culminate in cirrhosis and hepatocellular carcinoma (HCC). Precise diagnosis and staging of hepatic fibrosis are essential to prevent liver cirrhosis and HCC. Simultaneous detection of multiplex collagen biomarkers within liver tissue is crucial for staging hepatic fibrosis. We herein for the first time constructed multiplex collagen fingerprinting for the staging of hepatic fibrosis using high-precision fluorescence-guided surface-enhanced Raman scattering (SERS) imaging. SERS/fluorescent probes, collectively referred to as SF, comprising silver nanoparticles (Ag NPs), Raman reporters, and FAM-labeled collagen targeting peptides. These probes exhibit exceptional aqueous dispersion and stability, attributed to the increased number of Asp residues in CTP. Meanwhile, SF probes, namely SF-I, SF-IV, and SF-D have demonstrated specific targeting of type I, type IV, and denatured collagen, respectively, within hepatic fibrotic tissues. The results from fluorescence-guided SERS imaging underscore the method's capacity for typing, localization, and quantification of collagen, thus providing novel insights into collagen's role in the development of hepatic fibrosis. The collagen fingerprinting strategy offers a potent toolkit for the multifaceted profiling of collagen superfamilies, holding significant implications for the precise staging of hepatic fibrosis.

Abstract B027: A blood-based, multiplex protein biomarker signature shows superior performance in detection of early-stage pancreatic ductal adenocarcinoma (PDAC)

Abstract BACKGROUND: A sensitive and specific blood biomarker test is needed to improve survival in PDAC through early detection. An initial discovery study analyzing more than 3000 proteins using both pre-selected immunoassays and the Olink proteomics platform identified 15 promising protein candidates, of which ten were successfully pre-verified for ELISA immunoassay analysis. This model-development study was conducted to develop and pre- validate a specific and sensitive biomarker signature for the early detection of PDAC. METHODS: This multicenter case-control study was performed using existing bio-banked pretreatment samples from patients with newly diagnosed PDAC (Stages I-II) and participants in PDAC high-risk surveillance programs as controls. Blinded quantitative measurement of protein biomarkers was performed using ELISA-based immunoassays. CA19-9 was measured on a Roche COBAS instrument. An AI-derived predictive mathematical biomarker signature incorporating multiple protein biomarkers was developed and assessed using standard cross- validation techniques. RESULTS: 128 Stage 1 and 2 PDACs and 495 high-risk control serum samples were analyzed with 10 predetermined biomarkers to develop a four-protein biomarker signature. The model predicted PDAC versus non-PDAC with 84% sensitivity at 98% specificity. Model performance was significantly better when compared to CA19-9 alone (sensitivity 65% at 98% specificity, p&amp;lt;0.001). In those patients with low CA19-9 (&amp;lt;37 U/ml), the model predicted PDAC with 62% sensitivity at 98% specificity, performing significantly better than CA19-9 alone (9% sensitivity at similar 98% specificity (p&amp;lt;0.001). Additionally, in patients &amp;gt;65 years old, the signature predicted PDAC with 90% sensitivity at 98% specificity, which was significantly more sensitive than CA19-9 alone (75% sensitivity at 98% specificity, p&amp;lt;0.001). Test accuracy was not affected by diabetes status. CONCLUSIONS: This blood-based, multiplexed biomarker signature showed high sensitivity and specificity and significantly outperformed CA19-9 for early-stage PDAC. This biomarker model has the potential to improve outcomes in PDAC patients through early detection. Analytical and clinical validation studies are planned. Citation Format: Norma Palma, Ralph Schiess, Thomas King, Alcibiade Athanasiou, Natasha Kureshi, Lisa Ford, Aimee Lucas, Bryson Katona, Randall Brand, Diane Simeone. A blood-based, multiplex protein biomarker signature shows superior performance in detection of early-stage pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B027.

Enhanced sensitivity in acute myocardial infarction diagnosis through optimized multiplex biomarker detection on a paper-based microfluidic platform with individual control capabilities

Acute myocardial infarction (AMI) is a severe condition with high mortality, requiring timely diagnosis, especially in low-resource settings where centralized testing is unavailable. Leveraging our previously developed, reliable, highly integrated on-chip rotary valving system on the microfluidic paper-based analytical device (µPAD), we propose a novel multiplex paper-based microfluidics immunoassay platform for the onsite detection of key cardiac biomarkers—Myoglobin (Myo), cardiac troponin I (cTnI), and creatine kinase-MB (CKMB)—in artificial serum. It enhances assay performance through individually controlled assay conditions, facilitating rapid AMI diagnosis. Compared to existing paper-based multiplex microfluidic ELISA systems, our µPAD uniquely enables the assay of each biomarker under separate, optimally controlled incubation conditions within a single test through the valving system, which leads to optimized signal-to-noise ratio (SNR) performance and introduces more flexibility in multiplex testing. The valving system in the µPAD is driven by a single servo motor, which is controlled via a microcontroller and an interactive touchscreen. These components are integrated into a highly programmable, easy-to-assemble platform that offers fast, low-cost, and user-friendly operation. Utilizing our platform, autonomous ELISA assays were performed on Myo, cTnI, and CKMB for calibration, achieving limits of detection (LOD) of 1.43 ng/mL for Myo, 0.2 ng/mL for cTnI, and 0.735 ng/mL for CKMB. Subsequent spike-and-recovery experiments also confirmed the platform's accuracy and precision in obtaining the test results within 30 minutes and 20 μL serum sample. These results highlight the platform's transformative potential for clinical AMI diagnosis and commercialization, providing a cost-effective and accessible solution for point-of-care testing in diverse settings.

Integrated biosensor array for multiplex biomarkers cancer diagnosis via in-situ self-assembly carbon nanotubes with an ordered inverse-opal structure

To enhance the precision and reliability of early disease detection, especially in malignancies, an exhaustive investigation of multi-target biomarkers is essential. In this study, an advanced integrated electrochemical biosensor array that demonstrates exceptional performance was constructed. This biosensor was developed through a controllable porous-size mechanism and in-situ modification of carbon nanotubes (CNTs) to quantify multiplex biomarkers—specifically, C-reaction protein (CRP), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA)—in human serum plasma. The fabrication process involved creating a highly ordered three-dimensional inverse-opal structure with the CNTs (pCNTs) modifier through microdroplet-based microfluidics, confined spatial self-assembly of nanoparticles, and chemical wet-etching. This innovative approach allowed for direct in-situ modification of nanomaterial onto the surface of electrode array, eliminating secondary transfer and providing exceptional control over structure and stability. The outstanding electrochemical performance was achieved through the synergistic effect of the pCNTs nanomaterial, aptamer, and horseradish peroxidase-labeled (HRP-) antibody. Additionally, the integrated biosensor array platform comprised multiple individually addressable electrode units (n = 11), enabling simultaneous multi-parallel/target testing, thereby ensuring accuracy and high throughput. Crucially, this integrated biosensor array accurately quantified multiplex biomarkers in human serum, yielding results comparable to commercial methods. This integrated technology holds promise for point-of-care testing (POCT) in early disease diagnosis and biological analysis.

Enzymatic Precipitation of Highly Electroactive and Ion-Transporting Prussian Blue for a Sensitive Electrochemical Immunosensor.

Sensitive and/or multiplex electrochemical biosensors often require efficient (bio)catalytic conversion of substrates into insoluble electroactive products. The enzymatic formation and precipitation of coordination polymers under mild conditions offers a promising solution for this purpose. Herein, we report the enzymatic precipitation of Prussian blue (PB), a highly electroactive and ion-transporting coordination polymer, on an immunosensing electrode for application in a sensitive electrochemical immunosensor for detecting thyroid-stimulating hormone (TSH). Five pairs of redox enzymes and their specific reductants were examined to achieve rapid PB precipitation and electrochemical oxidation. Among these pairs, O2-insensitive flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) paired with glucose yielded the highest electrochemical signal-to-background (S/B) ratio. FAD-GDH catalyzed the conversion of Fe(CN)63- to Fe(CN)64-, which coordinated with Fe3+, leading to PB formation and subsequent precipitation through repeated conversions. The resulting PB precipitate, with its close proximity to the electrode, facilitated rapid electrochemical oxidation and generated a strong electrochemical signal. Notably, the precipitation and electrochemical oxidation of PB were more effective than those of its analogues. When applied to a sandwich-type immunosensor for TSH detection, the enzymatic PB precipitation achieved a calculated detection limit of approximately 2 pg/mL in artificial serum, covering the clinically relevant range. These findings indicate the potential widespread utility of PB precipitation and electrochemical oxidation for sensitive multiplex biomarker detection.

Pathologic response rates to neoadjuvant pembrolizumab in locally advanced (LA) resectable cutaneous squamous cell carcinoma (cSCC).

9591 Background: Cutaneous squamous cell carcinoma (cSCC) is the second commonest cutaneous malignancy, accounting for 20% of cutaneous malignancies and 75% of non-melanoma skin cancer deaths. Anti-PD-1 is approved in unresectable and metastatic cSCC, and is associated with high rates of pathologic response in resectable locally advanced (LA) cSCC. We conducted a phase II single-arm neoadjuvant trial of pembrolizumab (P) in patients with PD-1 naïve resectable LA cSCC. Methods: Patients with AJCC/UICC ≥T3 (or T2 with ≥2 BWH high-risk features) and/or N+ disease were eligible. Patients received 2 cycles of P (200mg Q3W) prior to definitive surgery, and 15 cycles post-surgery (NCT04808999). The primary endpoint was pathologic complete response (pCR, defined as 0% residual viable tumor, RVT) per independent central pathology review. Key secondary endpoints included incidence of adverse events (AEs), recurrence-free survival (RFS), overall survival (OS), safety and unbiased spatial biomarkers. A selected panel of multiplex immunofluorescence imaging biomarkers were used by PredxBio Inc.’s computational pipeline to reveal spatial intratumor heterogeneity in the tumor microenvironment (TME). Results: Thirty patients were enrolled and received at least 1 cycle of P. The median age was 79 (59-93) years and patients were predominantly male (77%). One patient withdrew consent prior to surgery. Three patients died prior to definitive surgery: 2 from COVID-19, a third from a NSTEMI possibly related to P. Of the 26 response-evaluable patients, AJCC/UICC stage at baseline was high-risk T2, T3, N1, and N2 in 2 (8%), 12 (46%), 9 (34.5%), and 3 (11.5%) patients, respectively. We observed pCR in 15 (57%), pathologic partial response (pPR, 10% &lt; %RVT &lt;50%) in 2 (8%) and pathologic non-response (pNR, &gt;50%RVT) in 8 (31%) patients. One patient is pending surgery. Postoperative radiotherapy (RT) was de-escalated in 54% of the patients (14/15 in pCR). Median RFS was 13 (pCR) versus 10.5 (non-pCR) months. One Grade 5 (NSTEMI) and two Grade 3 (hepatitis, colitis) treatment-related AEs were observed. Computational analysis identifies key spatial interactions between PDL-1+/CD68+ cells implicated in response to P. Conclusions: Neoadjuvant P is efficacious in resectable LA cSCC with a high pCR rate (57%). Three deaths were observed, including 2 unrelated to treatment and 1 possible Grade 5 cardiac irAE, reflecting the inherent risks of perioperative therapy in this patient population. RT was de-escalated in 93% of pCR patients. No relapse events in pCR patients. Spatial interactions between PDL-1+ cells and CD68+ macrophages have a role in therapeutic response and will be detailed along with other spatial analyses of the TME compartment that have an impact on outcomes. Clinical trial information: NCT04808999 .

Development of an ultra-sensitive multiplex cytokine assay utilizing a novel digital multi-omics detection technology

Abstract Demand for rapid, highly sensitive measurement of proteins and nucleic acids has increased dramatically over the past several years to support biomarker research, drug development and diagnostics. In addition, the ability to interrogate both protein and nucleic acids (multi-omics) in a single sample allows critical information to be obtained relating the transcriptome to expressed protein levels. Despite this, current platforms for multiplex measurement of circulating biomarkers can lack sensitivity and specificity required for analytical and clinical validation of low abundance proteins and nucleic acids. We report the development of an automated microfluidic-based technology capable of multiplexed measurement of protein and nucleic acid biomarkers utilizing a novel approach combining digital and analog detection modalities. Significant improvements in sensitivity and specificity are achieved through use of a bead-based microfluidic array resulting in a spatially-resolved series of single-plex signal amplification reactions. Critically, the isolation of reactions allows multiplex panels to be constructed in a modular manner while eliminating interference that often limits specificity of traditional multiplex biomarker detection technologies. A specific application of this technology we report here is a multiplexed assay measuring low abundance cytokine biomarkers from serum and plasma samples with utility in a broad range of research and clinical studies.

Carbon nanotubes integrated photonic barcodes in Herringbone Microfluidics for Multiplex Biomarker Quantification

Early monitoring of cardiovascular disease (CVD) is crucial for its treatment and prognosis. Hence, highly specific and sensitive detection method is urgently needed. In this study, we propose a novel herringbone microfluid chip with aptamer functionalized core-shell photonic crystal (PhC) barcode integration for high throughput multiplex CVD detection. Based on the PhC derived from co-assembled carboxylated single-wall carbon nanotubes and silicon dioxide nanoparticles, we obtain core-shell PhC barcodes by hydrogel replicating and partially etching. These core-shell PhC barcodes not only retain the original structural colors coding element, but also fully expose a large number of carboxyl elements in the ore for the probe immobilization. We further combine the functionalized barcodes with herringbone groove microfluidic chip to elucidate its acceptability in testing clinical sample. It is demonstrated that the special design of microfluidic chip can significantly enhance fluid vortex resistance and contact frequency, improving the sample capture efficiency and detection sensitivity. These features indicate that our core-shell PhC barcodes-integrated herringbone microfluidic system possesses great potential for multiplex biomarker detection in clinical application.

Combining Blood-Based Biomarkers and Structural MRI Measurements to Distinguish Persons with and without Significant Amyloid Plaques.

Amyloid-β (Aβ) plaques play a pivotal role in Alzheimer's disease. The current positron emission tomography (PET) is expensive and limited in availability. In contrast, blood-based biomarkers (BBBMs) show potential for characterizing Aβ plaques more affordably. We have previously proposed an MRI-based hippocampal morphometry measure to be an indicator of Aβ plaques. To develop and validate an integrated model to predict brain amyloid PET positivity combining MRI feature and plasma Aβ42/40 ratio. We extracted hippocampal multivariate morphometry statistics from MR images and together with plasma Aβ42/40 trained a random forest classifier to perform a binary classification of participant brain amyloid PET positivity. We evaluated the model performance using two distinct cohorts, one from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and the other from the Banner Alzheimer's Institute (BAI), including prediction accuracy, precision, recall rate, F1 score, and AUC score. Results from ADNI (mean age 72.6, Aβ+ rate 49.5%) and BAI (mean age 66.2, Aβ+ rate 36.9%) datasets revealed the integrated multimodal (IMM) model's superior performance over unimodal models. The IMM model achieved prediction accuracies of 0.86 in ADNI and 0.92 in BAI, surpassing unimodal models based solely on structural MRI (0.81 and 0.87) or plasma Aβ42/40 (0.73 and 0.81) predictors. Our IMM model, combining MRI and BBBM data, offers a highly accurate approach to predict brain amyloid PET positivity. This innovative multiplex biomarker strategy presents an accessible and cost-effective avenue for advancing Alzheimer's disease diagnostics, leveraging diverse pathologic features related to Aβ plaques and structural MRI.

Visual and quantitative lateral flow immunoassay based on Au@PS SERS tags for multiplex cardiac biomarkers

Rapid and sensitive detection of multiple biomarkers by lateral flow immunoassay (LFIA) remains challenging for signal amplification for commonly used nanotags. Herein, we report a novel LFIA strip for visual and highly sensitive analysis of two cardiac biomarkers based on functionalized gold nanoparticles @ polystyrene microsphere (Au@PS）microcavity as surface-enhanced Raman scattering (SERS) tags. Antibody-modified Au@PS was designed as a SERS label. The evanescent waves propagating along the surface of the PS microcavity and the localized surface plasmons of the gold nanoparticles were coupled to enhance the light-matter interaction synergistically for Raman signal enhancement. In this strategy, the proposed Au@PS SERS tags-based LFIA was carried out to quantify the content of the heart failure and infarct biomarkers synchronously within 15 min and get the limits of detection of 1 pg/mL and 10 pg/mL for cardiac troponin I (cTnI) and N-terminal natriuretic peptide precursor (NT-proBNP), respectively. The results demonstrated 10–20 folds more sensitivity than that of the standard colloidal gold strip and fluorescent strip for the same biomarkers. This novel quantitative LFIA shows promise as a high-sensitive and visual sensing method for relevant clinical and forensic analysis.

Comparison between next-generation sequencing and multiplex polymerase chain reaction assays for nonsmall-cell lung cancer molecular diagnosis.

Genetic mutation detection has become an important step in nonsmall-cell lung cancer (NSCLC) treatment because of the increasing number of drugs that target genomic rearrangements. A multiplex test that can detect multiple gene mutations prior to treatment is thus necessary. Currently, either next-generation sequencing (NGS)-based or polymerase chain reaction (PCR)-based tests are used. We evaluated the performance of the Oncomine Dx Target Test (ODxTT), an NGS-based multiplex biomarker panel test, and the AmoyDx Pan Lung Cancer PCR Panel (AmoyDx PLC panel), a real-time PCR-based multiplex biomarker panel test. Patients with histologically diagnosed NSCLC and a sufficient sample volume to simultaneously perform the AmoyDx PLC panel and ODxTT-M were included in the study. The success and detection rates of both tests were evaluated. Biopsies revealed 116 cases of malignancies, 100 of which were NSCLC. Of these, 59 met the inclusion criteria and were eligible for analysis. The success rates were 100% and 98% for AmoyDx PLC panel and ODxTT-M, respectively. Nine driver mutations were detected in 35.9% and 37.3% of AmoyDx PLC and ODxTT-M panels, respectively. EGFR mutations were detected in 14% and 12% of samples using the AmoyDx PLC panel and ODxTT-M, respectively. Of the 58 cases in which both NGS and AmoyDx PLC panels were successful, discordant results were observed in seven cases. These differences were mainly due to different sensitivities of the detection methods used and the gene variants targeted in each test. The AmoyDx PLC panel, a PCR-based multiplex diagnostic test, exhibits a high success rate. The frequency of the nine genes targeted for treatment detected by the AmoyDx PLC panel was comparable to the frequency of mutations detected by ODxTT-M. Clinicians should understand and use the AmoyDx PLC panel and ODxTT-M with respect to their respective performances and limitations.

Simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues by multicolor quantum dots-based peptide probes

The accurate detection of multiplex collagen biomarkers is vital for diagnosing and treating various critical diseases such as tumors and fibrosis. Despite the attractive optical properties of quantum dots (QDs), it remains technically challenging to create stable and specific QDs-based probes for multiplex biological imaging. We report for the first time the construction of multi-color QDs-based peptide probes for the simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues. A bipeptide system composed of a glutathione (GSH) host peptide and a collagen-targeting guest peptide (CTP) has been developed, yielding CTP-QDs probes that exhibit exceptional luminescence stability when exposed to ultraviolet irradiation and mildly acidic conditions. The versatile bipeptide system allows for facile one-pot synthesis of high-quality multicolor CTP-QDs probes, exhibiting superior selectivity in targeting critical collagen biomarkers including denatured collagen, type I collagen, type II collagen, and type IV collagen. The multicolor CTP-QDs probes have demonstrated remarkable efficacy in simultaneously fingerprinting multiple collagen types in diverse connective tissues, irrespective of their status, whether affected by injury, diseases, or undergoing remodeling processes. The innovative multicolor CTP-QDs probes offer a robust toolkit for the multiplex fingerprinting of the collagen suprafamily, demonstrating significant potential in the diagnosis and treatment of collagen-related diseases.

Plasma proteins and persistent postsurgical pelvic pain among adolescents and young adults with endometriosis

Noninvasive biomarkers that predict surgical treatment response would inform personalized treatments and provide insight into potential biologic pathways underlying endometriosis-associated pain and symptom progression. To use plasma proteins in relation to the persistence of pelvic pain following laparoscopic surgery in predominantly adolescents and young adults with endometriosis using a multiplex aptamer-based proteomics biomarker discovery platform. We conducted a prospective analysis including 142 participants with laparoscopically-confirmed endometriosis from the Women's Health Study: From Adolescence to Adulthood observational longitudinal cohort with study enrollment from 2012-2018. Biologic samples and patient data were collected with modified World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project tools. In blood collected before laparoscopic ablation or excision of endometriosis, we simultaneously measured 1305 plasma protein levels, including markers for immunity, angiogenesis, and inflammation, using SomaScan. Worsening or persistent postsurgical pelvic pain was defined as having newly developed, persistent (ie, stable), or worsening severity, frequency, or persistent life interference of dysmenorrhea or acyclic pelvic pain at 1-year postsurgery compared with presurgery. We calculated odds ratios and 95% confidence intervals using logistic regression adjusted for age, body mass index, fasting status, and hormone use at blood draw. We applied Ingenuity Pathway Analysis and STRING analysis to identify pathophysiologic pathways and protein interactions. The median age at blood draw was 17 years (interquartile range, 15-19 years), and most participants were White (90%). All had superficial peritoneal lesions only and were treated by excision or ablation. One-year postsurgery, pelvic pain worsened or persisted for 76 (54%) of these participants with endometriosis, whereas pelvic pain improved for 66 (46%). We identified 83 proteins associated with worsening or persistent pelvic pain 1-year postsurgery (nominal P<.05). Compared with those with improved pelvic pain 1-year postsurgery, those with worsening or persistent pelvic pain had higher plasma levels of CD63 antigen (odds ratio, 2.98 [95% confidence interval, 1.44-6.19]) and CD47 (odds ratio, 2.68 [95% confidence interval, 1.28-5.61]), but lower levels of Sonic Hedgehog protein (odds ratio, 0.55 [95% confidence interval, 0.36-0.84]) in presurgical blood. Pathways related to cell migration were up-regulated, and pathways related to angiogenesis were down-regulated in those with worsening or persistent postsurgical pelvic pain compared with those with improved pain. When we examined the change in protein levels from presurgery to postsurgery and its subsequent risk of worsening or persistent postsurgical pain at 1-year follow-up, we observed increasing levels of Sonic Hedgehog protein from presurgery to postsurgery was associated with a 4-fold increase in the risk of postsurgical pain (odds ratio [quartile 4 vs 1], 3.86 [1.04-14.33]). Using an aptamer-based proteomics platform, we identified plasma proteins and pathways associated with worsening or persistent pelvic pain postsurgical treatment of endometriosis among adolescents and young adults that may aid in risk stratification of individuals with endometriosis.

Deep-learning assisted zwitterionic magnetic immunochromatographic assays for multiplex diagnosis of biomarkers

Magnetic nanoparticle (MNP)-based immunochromatographic tests (ICTs) display long-term stability and an enhanced capability for multiplex biomarker detection, surpassing conventional gold nanoparticles (AuNPs) and fluorescence-based ICTs. In this study, we innovatively developed zwitterionic silica-coated MNPs (MNP@Si-Zwit/COOH) with outstanding antifouling capabilities and effectively utilised them for the simultaneous identification of the nucleocapsid protein (N protein) of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) and influenza A/B. The carboxyl-functionalised MNPs with 10% zwitterionic ligands (MNP@Si-Zwit 10/COOH) exhibited a wide linear dynamic detection range and the most pronounced signal-to-noise ratio when used as probes in the ICT. The relative limit of detection (LOD) values were achieved in 12 min by using a magnetic assay reader (MAR), with values of 0.0062 ng/mL for SARS-CoV-2 and 0.0051 and 0.0147 ng/mL, respectively, for the N protein of influenza A and influenza B. By integrating computer vision and deep learning to enhance the image processing of immunoassay results for multiplex detection, a classification accuracy in the range of 0.9672–0.9936 was achieved for evaluating the three proteins at concentrations of 0, 0.1, 1, and 10 ng/mL. The proposed MNP-based ICT for the multiplex diagnosis of biomarkers holds substantial promise for applications in both medical institutions and self-administered diagnostic settings.