The development of integrated instrumentation for universal bioassay systems serves as a key goal for the lab-on-a-chip community. The programmable bio-nano-chip (p-BNC) system is a versatile ...multiplexed and multiclass chemical- and bio-sensing system for bioscience and clinical measurements. The system is comprised of two main components, a disposable cartridge and a portable analyzer. The customizable single-use plastic cartridges, which now can be manufactured in high volumes using injection molding, are designed for analytical performance, ease of use, reproducibility, and low cost. These labcard devices implement high surface area nano-structured biomarker capture elements that enable high performance signaling and are index-matched to real-world biological specimens. This detection modality, along with the convenience of on-chip fluid storage in blisters and self-contained waste, represents a standard process to digitize biological signatures at the point-of-care. A companion portable analyzer prototype has been developed to integrate fluid motivation, optical detection, and automated data analysis, and it serves as the human interface for complete assay automation. In this report, we provide a systems-level perspective of the p-BNC universal biosensing platform with an emphasis on flow control, device integration, and automation. To demonstrate the flexibility of the p-BNC, we distinguish diseased and non-case patients across three significant disease applications: prostate cancer, ovarian cancer, and acute myocardial infarction. Progress towards developing a rapid 7 minute myoglobin assay is presented using the fully automated p-BNC system.
As COVID-19 pandemic public health measures are easing globally, the emergence of new SARS-CoV-2 strains continue to present high risk for vulnerable populations. The antibody-mediated protection ...acquired from vaccination and/or infection is seen to wane over time and the immunocompromised populations can no longer expect benefit from monoclonal antibody prophylaxis. Hence, there is a need to monitor new variants and its effect on vaccine performance. In this context, surveillance of new SARS-CoV-2 infections and serology testing are gaining consensus for use as screening methods, especially for at-risk groups. Here, we described an improved COVID-19 screening strategy, comprising predictive algorithms and concurrent, rapid, accurate, and quantitative SARS-CoV-2 antigen and host antibody testing strategy, at point of care (POC). We conducted a retrospective analysis of 2553 pre- and asymptomatic patients who were tested for SARS-CoV-2 by RT-PCR. The pre-screening model had an AUC (CI) of 0.76 (0.73-0.78). Despite being the default method for screening, body temperature had lower AUC (0.52 0.49-0.55) compared to case incidence rate (0.65 0.62-0.68). POC assays for SARS-CoV-2 nucleocapsid protein (NP) and spike (S) receptor binding domain (RBD) IgG antibody showed promising preliminary results, demonstrating a convenient, rapid (<20 min), quantitative, and sensitive (ng/mL) antigen/antibody assay. This integrated pre-screening model and simultaneous antigen/antibody approach may significantly improve accuracy of COVID-19 infection and host immunity screening, helping address unmet needs for monitoring vaccine effectiveness and severe disease surveillance.
The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a “plug and play” ...approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of ≤102 different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10 000 and limit of detection values of ∼10-13 M are obtained readily with this microbead array system.
A novel library of resin-bound receptors within a cross-reactive differential array for the identification and discrimination of tripeptides and tripeptide mixtures is reported. Pattern recognition ...using principal component analysis showed complete discrimination of four similar tripeptides and three tripeptide mixtures. The library is comprised of a Cu(II)-centered core with two proximally appended tripeptide arms emanating outward. One tripeptide arm was prepared using combinatorial chemistry to generate the differential nature of the library. Thirty resin-bound receptors were randomly selected from the library and placed within a silicon microchip array that included integrated microfluidics elements, and an indicator-uptake assay was used for colorimetric signaling. The indicator Orange G yielded an accurate measure of the degree of association between receptors and analytes as determined by kinetic analysis of the indicator-uptake assays. Within this paper we detail the method used for differential sensing using a novel receptor library. This work further demonstrates the power and utility of a differential array of synthetic receptors for identification and discrimination of complex bioanalytes.
: Salivary secretions contain a variety of molecules that reflect important pathophysiological activities. Quantitative changes of specific salivary biomarkers could have significance in the ...diagnosis and management of both oral and systemic diseases. Modern point‐of‐care technologies with enhanced detection capabilities are needed to implement a significant advancement in salivary diagnostics. One such promising technology is the recently described lab‐on‐a‐chip (LOC) assay system, in which assays are performed on chemically sensitized beads populated into etched silicon wafers with embedded fluid handling and optical detection capabilities. Using this LOC system, complex assays can be performed with small sample volumes, short analysis times, and markedly reduced reagent costs. This report describes the use of LOC methodologies to assess the levels of interleukin‐1β (IL‐1β), C‐reactive protein (CRP), and matrix metalloproteinase‐8 (MMP‐8) in whole saliva, and the potential use of these biomarkers for diagnosing and categorizing the severity and extent of periodontitis. This study demonstrates that the results achieved by the LOC approach are in agreement with those acquired with standard enzyme‐linked immunosorbent assay (ELISA), with significant IL‐1β and MMP‐8 elevations in whole saliva of periodontitis patients. Furthermore, because of the superior detection capacities associated with the LOC approach, unlike those with ELISA, significant differences in CRP levels between periodontitis patients and normal subjects are observed. Finally, principal component analysis (PCA) is performed to yield an efficient method to discriminate between periodontally healthy and unhealthy patients, thus increasing the diagnostic value of these biomarkers for periodontitis when examined with the integrated LOC sensor system.
The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of “electronic taste chips” whereby multiple solution-phase ...analytes such as acids, bases, metal cations, and biological cofactors were detected and quantitated. The newly fashioned “cardiac chip” exploits a geometry that allows for isolation and entrapment of single polymeric spheres in micromachined pits while providing to each bead the rapid introduction of a series of reagents/washes through microfluidic structures. The combination of these miniaturized components fosters the completion of complex assays with short analysis times using small sample volumes. Optical signals derived from single beads are used to complete immunological tests that yield outstanding assay characteristics. The power and utility of this new methodology is demonstrated here for the simultaneous detection of the cardiac risk factors, C-reactive protein and interleukin-6, in human serum samples. This demonstration represents the first important step toward the development of a useful cardiac chip that targets numerous risk factors concurrently and one that can be customized readily for specific clinical settings.
•An accurate Multivariate Analytical Risk Index for Oral Cancer has been developed.•Accuracy ranged from 76.0–82.4–89.6% for benign, dysplastic, malignant lesions.•MARIO represents a new noninvasive ...tool to assist in disease monitoring of PMOL.
The diagnosis and management of oral cavity cancers are often complicated by the uncertainty of which patients will undergo malignant transformation, obligating close surveillance over time. However, serial biopsies are undesirable, highly invasive, and subject to inherent issues with poor inter-pathologist agreement and unpredictability as a surrogate for malignant transformation and clinical outcomes. The goal of this study was to develop and evaluate a Multivariate Analytical Risk Index for Oral Cancer (MARIO) with potential to provide non-invasive, sensitive, and quantitative risk assessments for monitoring lesion progression.
A series of predictive models were developed and validated using previously recorded single-cell data from oral cytology samples resulting in a “continuous risk score”. Model development consisted of: (1) training base classification models for each diagnostic class pair, (2) pairwise coupling to obtain diagnostic class probabilities, and (3) a weighted aggregation resulting in a continuous MARIO.
Diagnostic accuracy based on optimized cut-points for the test dataset ranged from 76.0% for Benign, to 82.4% for Dysplastic, 89.6% for Malignant, and 97.6% for Normal controls for an overall MARIO accuracy of 72.8%. Furthermore, a strong positive relationship with diagnostic severity was demonstrated (Pearson’s coefficient = 0.805 for test dataset) as well as the ability of the MARIO to respond to subtle changes in cell composition. The development of a continuous MARIO for PMOL is presented, resulting in a sensitive, accurate, and non-invasive method with potential for enabling monitoring disease progression, recurrence, and the need for therapeutic intervention of these lesions.
Background
The effective detection and monitoring of potentially malignant oral lesions (PMOL) are critical to identifying early‐stage cancer and improving outcomes. In the current study, the authors ...described cytopathology tools, including machine learning algorithms, clinical algorithms, and test reports developed to assist pathologists and clinicians with PMOL evaluation.
Methods
Data were acquired from a multisite clinical validation study of 999 subjects with PMOLs and oral squamous cell carcinoma (OSCC) using a cytology‐on‐a‐chip approach. A machine learning model was trained to recognize and quantify the distributions of 4 cell phenotypes. A least absolute shrinkage and selection operator (lasso) logistic regression model was trained to distinguish PMOLs and cancer across a spectrum of histopathologic diagnoses ranging from benign, to increasing grades of oral epithelial dysplasia (OED), to OSCC using demographics, lesion characteristics, and cell phenotypes. Cytopathology software was developed to assist pathologists in reviewing brush cytology test results, including high‐content cell analyses, data visualization tools, and results reporting.
Results
Cell phenotypes were determined accurately through an automated cytological assay and machine learning approach (99.3% accuracy). Significant differences in cell phenotype distributions across diagnostic categories were found in 3 phenotypes (type 1 “mature squamous”, type 2 “small round”, and type 3 “leukocytes”). The clinical algorithms resulted in acceptable performance characteristics (area under the curve of 0.81 for benign vs mild dysplasia and 0.95 for benign vs malignancy).
Conclusions
These new cytopathology tools represent a practical solution for rapid PMOL assessment, with the potential to facilitate screening and longitudinal monitoring in primary, secondary, and tertiary clinical care settings.
A point‐of‐care oral cytology tool has been developed for the noninvasive detection and monitoring of potentially malignant oral lesions. The distribution of cell phenotypes identified by machine learning and a cytology‐on‐a‐chip approach provides useful information as part of the assessment of oral lesions, with improved interpretability, calibration, and generalizability compared with conventional methods.
This perspective highlights the major challenges for the bioanalytical community, in particular the area of lab-on-a-chip sensors, as they relate to point-of-care diagnostics. There is a strong need ...for general-purpose and universal biosensing platforms that can perform multiplexed and multiclass assays on real-world clinical samples. However, the adoption of novel lab-on-a-chip/microfluidic devices has been slow as several key challenges remain for the translation of these new devices to clinical practice. A pipeline of promising medical microdevice technologies will be made possible by addressing the challenges of integration, failure to compete with cost and performance of existing technologies, requisite for new content, and regulatory approval and clinical adoption.
PDF
