A Label-free Optical Biosensor-Based Point-of-Care Test for the Rapid Detection of Monkeypox Virus.

Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (∼3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.

A spatially uniform illumination source for widefield multi-spectral optical microscopy.

Illumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifies the source field distribution to compensate for spatial non-uniformity introduced by optical components in the imaging system. In this study, we characterize the light coupling efficiency of the proposed design and compare the uniformity performance with the conventional method. EUCLID demonstrates a remarkable illumination improvement for multi-spectral imaging in both Nelsonian and Koehler alignment with a maximum spatial deviation of ≈ 1% across a wide field-of-view.

Attomolar sensitivity microRNA detection using real-time digital microarrays.

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to [Formula: see text] 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.

Sensitive and real-time detection of IgG using interferometric reflecting imaging sensor system.

Considering the limitations of well-known traditional detection techniques, innovative research studies have focused on the development of new sensors to offer label-free, highly sensitive, real-time, low-cost, and rapid detection for biomolecular interactions. In this study, we demonstrate immunoglobulin G (IgG) detection in aqueous solutions by using real-time and label-free kinetic measurements of the Interferometric Reflectance Imaging Sensor (IRIS) system. By performing kinetic characterization experiments, the sensor's performance is comprehensively evaluated and a high correlation coefficient value (>0.94) is obtained in the IgG concentration range of 1-50 µg/mL with a low detection limit (0.25 µg/mL or 1.67 nM). Moreover, the highly sensitive imaging system ensures accurate quantification and reliable validation of recorded binding events, offering new perspectives in terms of direct biomarker detection for clinical applications.

Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements.

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have a higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in a 24-multiplexed chip format with less than 5% measurement error.