Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise ...improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC)-a multiplexable, microfluidic, modular platform-to quantify a novel multi-marker panel comprising CA125, HE4, MMP-7, and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4% and 10.5%, well below the biologic variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R(2) = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with prediagnostic plasma from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels.
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.
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.
The programmable bio-nano-chip (p-BNC) is an ultra-flexible system for multiplexed and multiclass assays on a universal modular lab-on-a-chip platform for clinical and bioscience applications at the point-of-care.
Abstract
The high mortality rate of ovarian cancer is directly related to the lack of an effective screening strategy, as up to 90% of cancers that have not metastasized beyond the ovaries can be ...treated effectively using current strategies. While the biomarker CA125 has been used with some success for detecting ovarian cancer, additional biomarkers can increase the sensitivity and specificity of an ovarian cancer diagnostic test. Here, a panel of 4 biomarkers, CA125, HE4, CA72-4 and MMP-7, has been multiplexed with the programmable Bio-Nano-Chip (p-BNC). A rapid, precise and multiplexable diagnostic system will more successfully validate these suspected biomarkers over traditional methods by utilizing less volume of precious clinical samples and significantly reducing assay times. Additionally, the p-BNC has potential to provide a method for the point-of-care analysis of patients’ health, reducing costs and turn-around times for results. This system utilizes antibodies on nano-nets of agarose microbeads to capture protein biomarkers for a fluorescence-based sandwich immunoassay. By housing the microbeads in individual addressable wells that allow for the sample and reagents to pass through the porous agarose, multiple biomarkers can be analyzed in a single assay. The in-development disposable microfluidic card is utilized to control sample and reagent processing and allows the fluid to interact with the beads, as well as contains all biowaste for safer disposal. Low limits of detection (LODs) and high precision have been achieved using the p-BNC with a short analysis time under an hour, confirming its ability to be used for measuring biomarker levels in the physiological rage. Dose response curves of the four individual biomarkers are performed on the p-BNC system and are compared to the multiplexed dose response curve to ensure minimal cross-reactivity between biomarker reagents. Additionally, all four biomarker assays are compared to EIA using late-stage clinical samples to validate the system's diagnostic value. Concurrently, samples from patients separated into blood, plasma and serum are being used to determine the feasibility of a finger-stick assay for the ovarian cancer biomarkers. A novel card for the p-BNC is now in development for analyzing finger-stick quantities of blood and is being demonstrated to work effectively with high concentrations as a proof of concept. The p-BNC shows strong promise to be an effective system capable of the multiplexed analysis of ovarian cancer biomarkers and now a retrospective trial in collaboration with MD Anderson and UKCTOCS is underway to create a risk of ovarian malignancy algorithm by utilizing pre-clinical sera from 50 patients destined to develop ovarian cancer.
Citation Format: Basil Shadfan, Archana Raamanathan, Glennon Simmons, Robert C. Bast, John T. McDevitt. Multiplexed detection of early-stage ovarian cancer biomarkers using a microfluidic bead-based immunosensor. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3495. doi:10.1158/1538-7445.AM2013-3495
Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large- scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise ...improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p- BNC) - a multiplexable, microfluidic, modular platform - to quantify a novel multimarker panel comprised of CA125, HE4, MMP-7 and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross- reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4-10.5% – well below the biological variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R2 = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex® platform. In a 31-patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with pre-diagnostic sera from 2 cases and 4 controls. An updated card capable of being manufactured on a large-scale was developed that enabled the transition to true point-of-care measurement. A custom backpack-sized analyzer capable of precise actuation of blister packs (attached to each card) was used to control fluid rates of the various stages and contains all hardware necessary for imaging and analysis. Without sacrificing analytical performance, the multiplexed assay was reduced to 17 minutes in order to return results during an average patient visit. In a 22-patient study, blood was shown to correlate well to plasma and serum in the p-BNC, demonstrating promise for whole blood fingerstick measurements. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening and takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels.
Abstract
Early detection of ovarian cancer could significantly impact clinical outcome and hence patient survival. Given the biological heterogeneity of ovarian cancer, multi-marker panels will be ...required. Validation of such panels must utilize pre-clinical serum samples that are available only in small quantities. Diagnostic platforms that are capable of rapid, precise and multiplexed analysis of biomarkers in small quantities of serum should facilitate validation of optimal biomarker panels. Once validated, these platforms could permit rapid screening with finger-stick quantities of blood at point-of-care. The Programmable Bio-Nano-Chip (p-BNC) microfluidic immunosensor provides such a platform. In the p-BNC, serum biomarkers are sequestered and assessed with a fluorescence-based sandwich immunoassay, completed in nano-nets of sensitized agarose microbeads localized in individually addressable wells, housed in a microfluidic module, capable of integrating multiple sample, reagent and biowaste processing and handling steps. Previously, we adapted the p-BNC for the quantification of the CA125 biomarker with low LODs, high precision and a short analysis time of 43 minutes. Here, we adapt the p-BNC to simultaneously assess CA125 and HE4, a promising biomarker panel implicated in early detection. The HE4 immunoassay was developed to perform optimally with the previously established CA125 assay as the rate determining step. Extensive cross reactivity analysis ruled out non-ideal interactions between HE4 and CA125 reagents at the level of capturing antibody, detecting antibody and individual analytes. Wash times were optimized to maximize p-BNC performance. The dose-response curves obtained for the individual analytes (singleplex) were similar to those obtained with multiplex assays. Identical LOD values were noted for the singleplex and multiplex immunoassays with 1.5-fold decrease in precision for the multiplexed assays, demonstrating negligible loss of analytical performance upon multiplexing. For clinical validation, 8 surgically confirmed advanced stage patient sera and 8 age-matched healthy controls were assessed on the multiplexed p-BNC. ROC curve analysis for the CA125-HE4 biomarker combination, interpreted with a Risk of Ovarian Malignancy Algorithm was able to discriminate diseased samples from healthy controls with an AUC of 1.00. The p-BNC indicates strong promise for robust, multiplexed, quantitative measurements of ovarian cancer biomarkers, with high precision and sensitivity to yield clinically pertinent information under 45 minutes. Incorporation of additional biomarkers is underway to establish and validate an optimal panel for early detection of ovarian cancer.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4067. doi:1538-7445.AM2012-4067
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