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Total Microfluidic chip for Multiplexed diagnostics (ToMMx)
Journal Pre-proof Total Microfluidic chip for multiplexed diagnostics (ToMMx) Mehmet O. Ozen, Kaushik Sridhar, Mehmet Giray Ogut, Akash Shanmugam, Anirudh S. Avadhani, Yukari Kobayashi, Joseph C. Wu, Francois Haddad, Utkan Demirci PII:
S0956-5663(19)31009-7
DOI:
https://doi.org/10.1016/j.bios.2019.111930
Reference:
BIOS 111930
To appear in:
Biosensors and Bioelectronics
Received Date: 24 July 2019 Revised Date:
12 November 2019
Accepted Date: 25 November 2019
Please cite this article as: Ozen, M.O., Sridhar, K., Ogut, M.G., Shanmugam, A., Avadhani, A.S., Kobayashi, Y., Wu, J.C., Haddad, F., Demirci, U., Total Microfluidic chip for multiplexed diagnostics (ToMMx), Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/j.bios.2019.111930. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
CRediT Author Statement Mehmet O. Ozen: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data Curation, Visualization, Writing – Original Draft. Kaushik Sridhar: Software, Formal Analysis, Writing – Review & Editing. Mehmet Giray Ogut: Visualization. Akash Shanmugam: Software, Resources. Anirudh S. Avadhani: Visualization. Yukari Kobayashi: Resources, Formal Analysis, Writing – Review & Editing. Joseph C. Wu: Supervision, Writing – Review & Editing. Francois Haddad: Resources, Formal Analysis, Writing – Review & Editing. Utkan Demirci: Conceptualization, Supervision, Writing – Review & Editing, Project Administration, Funding Acquisition.
Total Microfluidic chip for Multiplexed diagnostics (ToMMx) Mehmet O. Ozen a, b, Kaushik Sridhar a, Mehmet Giray Ogut a, Akash Shanmugam a, Anirudh S. Avadhani a, Yukari Kobayashi b, Joseph C. Wu b, c , Francois Haddad b, c and Utkan Demirci a, b * Affiliations: a
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early
Detection, Department of Radiology, School of Medicine Stanford University Palo Alto, CA 94304, US. b
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305,
US c
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305, US
* Corresponding author. Corresponding author’s e-mail: [email protected] (Utkan Demirci, Ph.D.)
Abstract Microfluidic technologies offer new platforms for biosensing in various clinical and point-of-care (POC) applications. Currently, at the clinical settings, the gold standard diagnostic platforms for multiplexed sensing are multi-step, time consuming, requiring expensive and bulky instruments with a constant need of electricity which makes them unsuitable for resource-limited or POC settings. These technologies are often limited by logistics, costly assays and regular maintenance.
Although there have been several attempts to miniaturize these diagnostic
platforms, they stand short of batch fabrication and they are dependent on complementary components such as syringe pumps. Here, we demonstrated the development and clinical testing of a disposable, multiplexed sensing device (ToMMx), which is a portable, high-throughput and user-friendly microfluidic platform. It was built with inexpensive plastic materials and operated manually without requiring electrical power and extensive training. We validated this platform in a small cohort of 50 clinical samples from patients with cardiovascular diseases and healthy controls. The platform is rapid and gives quantifiable results with high sensitivity, as low as 5.29 pg/mL, from only a small sample volume (4 µL). ToMMx platform was compared side-by-side with commercial ELISA kits where the total assay time is reduced 15-fold, from 5 hours to 20 minutes. This technology platform is broadly applicable to various diseases with well-known biomarkers in diagnostics and monitoring, especially with potential future impact at the POC settings.
Key words: Microfluidics, multiplexed sensing, on-chip manual biosensing, cardiovascular biomarkers
1. Introduction Blood tests are one of the standard tools used in clinic to diagnose various conditions and monitor diseases and the efficacy of therapeutic interventions (Gaggin and Januzzi, 2013; Ho et al., 2018; Huang et al., 2017; Mozaffarian et al., 2016; Omland and White, 2017; Vasan, 2006). The current biomarker detection systems; such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay or mass spectrometry, are accurate, well-established and sensitive assays. However, they require central laboratory settings and have limited potential for multiplexing. The cost is also a major limiting factor requiring significant human and material resources. Further, these assays are not suitable to satisfy the logistical and practical needs at point-of-care (POC) settings. POC devices have the advantage of sensing multiple biomarkers from a small volume of sample with shorter assay time compared to conventional assays (Verhertbruggen et al., 2009). To provide access to affordable POC monitoring, without trading off the ability to perform clinically relevant assays, we (Mani et al., 2016; Wang et al., 2014, 2011) and others (Adel Ahmed and Azzazy, 2013; Badu-Tawiah et al., 2015; Cheng et al., 2010; Fu et al., 2010; Mandon et al., 2010; Pollock et al., 2012; Sia et al., 2004; Sollier et al., 2009; Yu et al., 2013) have developed microfluidic biosensing platforms, based on specific capture and sensing of circulating biomarkers. However, none of these assays targeted multiple myocardial infarction and heart failure related biomarkers specifically and simultaneously. POC systems offer versatile, portable and easy-to-use platforms, which have lower production and analysis cost per sample, and eliminate requirements of trained personnel for application and interpretation of the resulting data (Drain et al., 2014). Developing a POC platform that may combine biomarkers from several mechanistic processes such as myocardial injury, wall stress, inflammation and oxidative stress in a simple cost-effective assay could improve care in resource scare areas.
Conventional POC diagnostic devices (e.g., lateral flow tests) have a wide-spread utility (Bruls et al., 2009; Drain et al., 2014), however, they generally can detect one or two biomarkers in series, and require compatible buffers and reagents for each target, where assays can crossreact and do not provide high sensitivity, precision, and quantitative results, making them unsuitable for cardiovascular biomarker detection (Carrio et al., 2015; Cho et al., 2009; Sher et al., 2017; Zhang et al., 2018). The advances in micro- and nano-technologies enable integration of POC diagnostic systems with well-established and sensitive quantitative assays (Krishnan et al., 2011; Tekin et al., 2013), such as ELISA, yielding miniaturized new POC platforms eliminating concerns with cross-reactivity (Bruls et al., 2009). However, widely used ELISA strategies need well-resourced clinical settings, and they are multi-step and can be labor intensive. Microfluidic technologies hold advantages over conventional platforms via lab-onchip applications to detect multiple biotargets from a small volume of biological samples at a relatively shorter assay time (Beyazkilic et al., 2016; Bruls et al., 2009; Dittmer et al., 2010; Laksanasopin et al., 2015; Ng et al., 2018, 2017; Piraino et al., 2016; Pollock et al., 2012; Sonawane et al., 2017; Song et al., 2012). The application of these principles to ELISA resulted in development of platforms for detection of various targets, including; cells, proteins, antibodies and lipids (Bruls et al., 2009; M. Cornaglia et al., 2014a, 2014b; Dittmer et al., 2010; Mani et al., 2016, 2011; Tekin et al., 2013; Wang et al., 2014; Woolley and Hayes, 2015). Even though there have been substantial proof-of-concept studies conducted, there is still limited number of studies performed for validation of cardiac biomarker sensing platforms, using actual clinical samples; and a translation-ready, pump- and power-free, clinical device for monitoring biomarkers in circulation has yet to be demonstrated.
Here, we developed a new platform for multiplexed detection of biomarkers from a small volume of plasma using rapid, portable, highly sensitive, quantitative, Total Microfluidics for Multiplexed diagnostics (ToMMx) platform. Then, we validated this platform by comparing it side-by-side with commercial ELISA kits evaluating clinical samples. This platform can be operated manually without sophisticated training, giving results within 20 min as the simplest and fastest manual ELISA on-a-chip ever reported for multiplexed biomarker detection. The platform is designed for simplicity so it can be produced using disposable and inexpensive plastic materials. To validate the system with clinical samples, we used cardiac troponin I (cTnI), heart type fatty acid binding protein (hFABP) and N-terminal pro B-type natriuretic peptide (NTproBNP), which are released from damaged and/or stressed cardiomyocytes, representing myocardial infarction and heart failure. These are chosen as model target molecules to validate the ToMMx platform (Gaggin and Januzzi, 2013; Omland and White, 2017). For optimal clinical utility, the platform has been optimized to have low and clinically relevant limit of detection (LOD) values, which were calculated as; 9.56 pg/mL, 92.5 pg/mL and 5.29 pg/mL for cTnI, hFABP and NT-proBNP, respectively in plasma. We used bio-banked samples collected from individuals
with
cardiovascular
diseases
(acute
coronary
syndrome
(ACS),
dilated
cardiomyopathy (DCM) and severe symptomatic artic stenosis patients undergoing transcatheter aortic valve replacement (AS)) and healthy controls. 2. Materials and Methods Reagents, materials, platform fabrication, characterization, optimization, sample processing in comparison with commercial assays and statistical analysis of the results can be found in the Supplementary Information.
3. Results and discussion 3.1. Design and chemistry of ToMMx platform We designed an on-chip ELISA technology, Total Microfluidic chip for Multiplexed diagnostics (ToMMx) platform, utilizing antibody functionalized magnetic beads as “mobile substrates”, prepared from polymethyl methacrylate (PMMA) and double-sided adhesive (DSA) polyethylene terephthalate (PET) film layers, all confined to a footprint of 7 cm x 8 cm (Fig. 1A and Fig. S1). ToMMx is laser-cut from these plastic sheets of different thicknesses and assembled using adhesive film layers between each layer (Fig. 1B). ToMMx platform chips are designed to have four lanes, three for biomarker detection and one internal control lane, which gives us ability to simultaneously evaluate the same sample for the presence of three different cardiovascular disease (CVD)-related biomarkers (Fig. 1B). Cardiac troponin-I (cTnI), hearttype fatty acid binding protein (hFABP) and N-terminal pro-brain natriuretic peptide (NTproBNP) are chosen as model CVD biomarkers to represent multiplexed biomarker detection capability of ToMMx. Static substrates, such as 96-well plate surfaces, have been used for performing traditional ELISA as a well-established method, where samples and different reagents are introduced into microwells sequentially according to the detection strategy desired (e.g., direct, indirect or sandwich ELISA) (Hosseini et al., 2018). We adopted similar assay steps and modified for ToMMx platform as; (1) magnetic bead functionalization with capture antibodies, (2) sample preparation, (3) reagent and sample loading on ToMMx, (4) analyte capture on magnetic beads, (5) biotinylated secondary antibody labeling, (6) streptavidin conjugated enzyme labeling, (7) TMB substrate catalysis and (8) colorimetric detection on spectrophotometer (Fig. 1C). These steps (4-7) are performed on the disposable ToMMx chip with magnetic beads that are actuated
from one chamber to another, housing assay reagents, washing buffer and mineral oil. To create an iterative filling into the chambers without mixing, we took advantage of surface tension differences between mineral oil versus water-based solutions. Reagents and buffers were preloaded on chip before sample processing, in an orderly fashion, where, first, we loaded oil chambers, then washing buffer chambers, and finally reagent chambers at the end (Fig. 1C, step 3). An assay stage to house the chip and a magnet holder to actuate beads during the assay through the assay route was designed and assembled using PMMA and DSA layers (Fig. S2).
Fig. 1. Schematic of the Total Microfluidic chip for Multiplexed diagnostics (ToMMx) platform and designed assay protocol. (A) Polymethyl methacrylate (PMMA) and double-sided adhesive (DSA) polyethylene terephthalate (PET) film layers of ToMMx design. (B) Laser-cut and assembled ToMMx. (C) Bead functionalization, sample preparation and assay steps of ToMMx. (1) Functionalization of tosylactivated magnetic beads with analyte specific primary antibodies. (2) Sample dilution buffer, plasma sample and functionalized beads mixed in tube as sample preparation. (3) Assay reagents, buffers and sample loading on ToMMx. (4) Analyte in the sample captured on antibody functionalized beads. (5) Analyte-antibody complex labeled with biotinylated secondary antibody. (6) Streptavidin conjugated poly-HRP binding to antibody-antigen-antibody sandwich complex. (7) TMB substrate catalysis by polyHRP in the complex. (8) Evaluation of analyte concentration via color change in the sample after transferring the colored liquid to a 96-well plate, mixing with stop solution and reading in with a spectrophotometer.
3.2. Simulating magnetic bead movement on ToMMx platform To better understand the bead-magnetic field interactions, in ToMMx, under the magnetic field applied by the neodymium magnets, we ran simulations (Fig. S3A-B). A three-dimensional (3D) model of ToMMx is used to calculate the magnetic flux density distribution of the magnets in various chambers of the device (Table S1). The magnetic susceptibility of PMMA, water-based buffers/reagents and oil used on ToMMx is much smaller compared to that of magnetic beads, hence, we neglect their contribution to magnetic field in the medium and approximated the magnetic field to be uniform. Simulations show that the magnetic forces increase as the distance between the beads and permanent magnets decreases (Fig. S3C). We compared three commercial magnetic beads of different sizes (1, 1.05, 2.8 µm in diameter, Table S2), and simulated the behavior of beads in terms of magnetic force, height and velocity over time in a chip chamber. Based on the multi-slice numerical simulation of the threedimensional model, the magnetic flux density in the positive z axis (Bz) is shown to vary from ~ 0.35 T (at the bottom of the chambers) to ~ 0.18 T (at the top of the chambers). Using this data, we estimate the time, velocity and force experienced by the beads on ToMMx. Magnetic beads with the highest magnetic susceptibility (
) respond better in chambers. According to
the simulations, the process takes 10 seconds for a magnetic bead to travel from the top of the chamber to the bottom. Then, we displace the magnets to a position under the consecutive compartment, where the beads can be efficiently transferred to the next chamber, following the magnet through the oil barrier, for the next assay step (Fig. S3D-F).
3.3. Analytical performance of the assay We first optimized the assay steps on a 96-well plate format by spiking target proteins in PBS, and established the limit of detection (LOD) values calculated as 0.4 pg/mL, 400 pg/mL, 25 pg/mL for cTnI, hFABP and NT-proBNP, respectively (Fig. 2A-C). To achieve a lower background signal for plasma samples, we evaluated four different sample diluents on the designed assay (Fig. S4). We chose two commercial diluents according to their higher signal-tonoise ratio for spiked plasma samples, and lower background signals for unspiked plasma samples (Fig. S5). We spiked plasma with target proteins and diluted with these diluents (cTnI and hFABP spiked plasma diluted with Plasma Diluent, NT-proBNP spiked plasma diluted with General Diluent) to define LOD on the 96-well plate format. We, then, calculated these LOD values as 2.5 pg/mL, 2.5 pg/mL, 40 pg/ mL for cTnI, hFABP and NT-proBNP, respectively (Fig. 2D-F).
Fig. 2. Assay optimization, limit of detection (LOD) in PBS and plasma. Target molecules (cTnI, hFABP and NT-proBNP) were spiked into PBS supplemented with 2% bovine serum albumin (BSA) and 0.05% Tween20. LOD values of the platform for three biomarkers were measured as 0.4 pg/mL (1.5%), 400 pg/mL (1.5%), 25 pg/ mL (6.6%) for (A) cTnI, (B) hFABP and (C) NT-proBNP, respectively. In a similar fashion, target molecules (cTnI, hFABP and NT-proBNP) were spiked into healthy plasma samples and diluted with optimized assay diluents. LOD of the platform for three biomarkers were observed as 2.5 pg/mL (5.9%), 2.5 pg/mL (2.7%), 40 pg/ mL (3.7%) for (D) cTnI, (E) hFABP and (F) NT-proBNP, respectively (). These LOD values were obtained on the 96-well format of the assay and the optimized parameters were translated on the ToMMx chip platform. Pink areas represent the average signal from six control samples (n=3, ± SD). Relative standard deviations (RSD) are given in brackets.
To reduce the background signal from plasma, we ran optimization steps for ToMMx platform (Fig. S5). We obtained high correlation (R2 ≥ 0.92) in comparison to the commercial ELISA kits when we evaluated the standard biomarkers (Fig. S6). Using the calibration curves obtained from these studies, we calculated Limit of Detection (LOD) and Limit of Quantitation (LOQ) of commercial kits and ToMMx platform (Table S3). LOD values are obtained as 9.56, 92.5, 5.29 pg/mL (10.1% RSD) for cTnI, hFABP and NT-proBNP, where LOQ values are found to be 28, 290 and 16.04 pg/mL, respectively for ToMMx. These LOD levels are significantly lower (around three orders of magnitude) compared to earlier reported on-chip ELISA strategies (Adel Ahmed and Azzazy, 2013). 3.4. Analytical performance of the platform in comparison with commercial kits We demonstrated analytical performance of cardiac biomarker biosensing on ToMMx, using clinical samples. We tested 38 patient samples (n = 11, acute coronary syndrome, ACS; n = 19, severe symptomatic aortic stenosis, AS; n = 8, dilated cardiomyopathy, DCM) and 12 control samples in head-to-head comparison with three commercial ELISA kits from two suppliers. Patient samples were obtained from the Healthy Aging study where patients were screened with London School of Hygiene dyspnea and chest pain questionnaire, echocardiograms and vascular ultrasound. Control samples were withdrawn from healthy individuals without evidence of clinical or subclinical cardiovascular disease. In addition, these individuals did not have a history of inflammatory conditions, cancer or acute infection. As shown in Table S4, patients with AS were older compared to remaining individuals. Left ventricular systolic function was preserved in patients with ACS, while it was reduced in patients with AS, and remarkable reduced in individuals with DCM. In ACS, cTnI levels tend to increase due to myocyte cell damage, where cytosolic hFABP leaks to circulation becoming detectable as well (Bjurman et al., 2015; Kurz et
al., 2011; Lippi et al., 2013; Schulz et al., 2007; Zhou et al., 2014). NT-proBNP is being monitored to diagnose and prognose individuals with heart failure, since circulating NT-proBNP concentration increases due to the increased left ventricular load (Januzzi et al., 2006, 2005; Ozturk et al., 2011; Weber and Hamm, 2006; Yancy et al., 2013; Yeo et al., 2003). Using ToMMx, we were able to distinguish control samples from individuals, who had one of the following conditions; ACS, AS and/or DCM, significantly with high sensitivity (Fig. 3A-F). When we computed the obtained data to prepare the receiver operating curves (ROC) for each biomarker, the area under the curve (AUC) values were depicted as 0.904, 0.741, and 0.750 for cTnI, hFABP and NT-proBNP, respectively on ToMMx (Fig. 3G-I). We also analyzed the results separating the samples as “diseased” and “control” for the presence of these three biomarkers. The signals obtained from diseased samples had statistically higher signal when ToMMx was used compared to commercial kits (Fig. S7A-C). The signals obtained from control samples were not significantly different between two methods (Fig. S7D-F). We, also, analyzed these results using Bland-Altman method, where 3 patient samples for cTnI, 2 patient samples for hFABP and 3 patient samples for NT-proBNP, and 1 healthy sample for all biomarkers exceeded the 95% confidence level (±1.96 SD) (Fig. S8). Furthermore, we evaluated looked for the presence of; cTnI and hFABP in 11 ACS patients, NT-proBNP in 19 AS and 8 DCM patients. Among these tested samples, ToMMx resulted with higher signals for cTnI, hFABP and NT-proBNP (for AS) detection in comparison to commercial kits (Fig. 4A-C). For NT-proBNP on DCM patients, both ToMMx and commercial kits gave comparable results (Fig. 4D). When ToMMx was used, we reached to ~ 91% accuracy to identify ACS (via cTnI and hFABP), and ~95% accuracy to identify AS (via NT-proBNP). We were able to also identify all DCM patients using NT-proBNP as a diagnostic biomarker (Fig. 4E-H).
Fig. 3. ToMMx vs commercial ELISA kits. We evaluated the presence of cTnI, hFABP and NTproBNP in 38 patient samples in comparison to 12 healthy plasma samples using (A-C) ToMMx platform and (D-F) commercial ELISA kits. Mann-Whitney unpaired t-test used to analyze the results. ToMMx platform was able to distinguish patient samples from healthy samples, significantly. Results obtained from commercial ELISA kits were non-significant. ROC curves present AUC values for (G) cTnI, (H) hFABP, (I) NT-proBNP (n=3-4, ± SD).
Fig. 4. Disease specificity. We analyzed the patient plasma results obtained from ToMMx via cTnI and hFABP for MI (n = 11), and via NT-proBNP for TAVR (n = 19) and DCM (n = 8) identification. ToMMx significantly differentiated, (A) cTnI (p = 0.038) and (B) hFABP (p=0.033) presence for MI, and (C) NT-proBNP (p=0.0022) for TAVR detection, where (D) NTproBNP results for DCM were not significantly different compared to commercial ELISA kit results. Mann-Whitney paired t-test used to analyze the results. Bland-Altman analysis performed to analyze the difference between methods for (E) ACS – cTnI, (F) ACS – hFABP, (G) AS - NT-proBNP, and (H) DCM - NT-proBNP. The 95% confidence intervals on the mean is shown on the graphs with red-dotted lines as ± 1.96 SD (n = 3-4, ± SD).
Cardiac diseases are major adverse events, where the interaction of tissues and cells in the cardiac niche, necessitate multiple biomarker approaches to decode their interplay through disease progression. The complexity within this network makes CVDs complex disorders, which can be potentially better identified, exercising multiple biomarker monitoring approaches, rather than focusing on a single biomarker (Bayes-Genis and Ordonez-Llanos, 2015; Dhingra and Vasan, 2017; Doehner, 2012; Giannessi, 2011; Ho et al., 2018; Vasan, 2006; Wang et al., 2017). Standard cardiac biomarker testing in routine practice consists of blood collection, plasma or serum separation and testing samples either using an automated platform in a centralized laboratory or using a commercial ELISA kit. These approaches are expensive and time consuming for daily practices, requiring trained personnel and routine device maintenance. Some of these, over time, become inaccurate due to delayed maintenance of the large-scale equipment associated with high maintenance costs, given that many of these centralized labs are not wellfunded, especially in developing regions. Moreover, especially in resource-limited settings, centralized sample processing and evaluation can bring complications such as lost samples and results. Further, the need for a centralized laboratory can significantly deaccelerate and delay acquisition and dissemination of time sensitive clinical results, where the POC diagnostic tests are available. To eliminate dependence on electrical power on sample processing, we optimized a system for manual operation considering resource-limited setting where the electrical power shut-downs are common and hence, we did not intend to take an automation path with the system. The developed platform has a lower sample analysis cost and there is no ELISA-based portable platform for POC that we can compare our test against, that is commercially available from a cost perspective. However, manufacturing cost of the chip relies on multiple variables,
including the ordered components, their production methods and distance to manufacturing site, as well as transportation costs. We anticipate that our device can be ultimately produced at a low cost given that it can be batch-fabricated by injection molding. The cost of the plastic pieces of ToMMx, PMMA and DSA layers, can be produced at a cost as low as 10 cents, and buffers of the assay can be purchased for 10 cents. Antibodies and magnetic beads constitute the remaining ~ 95% of the total cost that can be potentially lowered by bulk orders for a commercial setting, at production scale workflows. Earlier magnetic bead-based ELISA systems have been reported by our lab and others (Adel Ahmed and Azzazy, 2013; Mani et al., 2016; Wang et al., 2014) however these systems did not present a multiplexed CVD biomarker sensing capability. Our approach presents a comprehensive study done in obtaining magnetic force of the magnetic beads to precisely estimate the time and velocity that should be followed to reach the best time and magnet movement in each chamber to achieve the optimum workflow, and hence, enhancing the assay performance. Minimizing / Eliminating sample preparation steps is one of the significant advantages of microfluidic platforms. Several strategies, that are either already commercialized or in the pipeline, utilizing fluid dynamics (Robinson et al., 2017), filtration (Tasso, n.d.), or micro needles (Blicharz et al., 2018), can be adapted to ToMMx platform easing the path for POC applications. This study focuses on the capability of an on-chip platform to perform multiplexed analysis resulting with clinically relevant LODs at minimal necessities. Current version of ToMMx is used in a laboratory environment evaluating plasma samples with the help of a spectrophotometer as a result enumeration strategy as a proof-of-concept study. With the further studies on buffers, bead concentrations and portable reading strategies ToMMx will be fully
adaptable for POC settings, minimizing assay steps such as using a stop solution, which we implemented in here to reduce the effect of sample transfer on the results. With further development and integration portable readers, this system has great potential for POC applications that would make the whole system more portable and easier to use eliminating the transfer steps and replacing the stop reactions with timed measurements (Inci et al., 2015; Liang et al., 2017; Wang et al., 2011). 4. Conclusion In this study, we designed a novel platform that combines the advantages of sensitivity, multiplexing, minimal sample volume, simplicity, low-cost and time effectiveness to sense multiple cardiac biomarkers simultaneously from a small volume of plasma sample. We validated the platform for three key cardiovascular biomarkers reflecting the pathway of myocardial injury (cTnI, h-FABP) and myocardial wall stress (NT-proBNP) in a small cohort of clinical samples. The whole assay can be performed manually by actuating magnetic beads with a simple off-the-shelf magnet without the requirement of a complex instrument. The quantitative results are precise and accurate compared to plate ELISA systems. Also, the total assay time is reduced 15-fold, with the help of reduced sample volume, increased assay surface area on magnetic beads, higher surface-to-volume ratio, and step-by-step compartmentalization of the assay on-chip. Developed platform performed well in sensitivity and specificity compared to commercial ELISA kits with significantly lower LOD values. This feature of our platform enabled us to detect targets within the clinically relevant cut-off concentrations (9.56 pg/mL for cTnI, and 5.29 pg/mL for NT-proBNP) only using a minute amount of a sample. Studies presented in this work establish the validation and clinical relevance, to further translate the tool into field studies.
In conclusion, we anticipate that our device will ultimately represent a laboratory-quality, multiplexed platform, utilizing the advantages of microfluidics to build a portable, highthroughput and user-friendly tool that can be performed by any individual with minimal training, simplifying and reducing the cost of diagnosis and monitoring of cardiovascular events. Conceivably, the development of this versatile, multiplex platform, with its flexible design, could facilitate the emergence of other clinically relevant POC assays with well-defined clinically important markers at multiple settings; including resource-limited settings, home, primary care, and bedside at the hospitals, after integration of ToMMx with portable readers, such as mobile phones.
CRediT authorship contribution statement Mehmet O. Ozen: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data Curation, Visualization, Writing – Original Draft.
Kaushik Sridhar:
Software, Formal Analysis, Writing – Review & Editing. Mehmet Giray Ogut: Visualization. Akash Shanmugam: Software, Resources. Anirudh S. Avadhani: Visualization. Yukari Kobayashi: Resources, Formal Analysis, Writing – Review & Editing. Francois Haddad: Resources, Formal Analysis, Writing – Review & Editing. Joseph C. Wu: Supervision, Writing – Review & Editing. Utkan Demirci: Conceptualization, Supervision, Writing – Review & Editing, Project Administration, Funding Acquisition.
Declaration of competing interest UD is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions, (iii) Hillel Inc., a company focused on cell phone-based assays. (iv) co-founder of and have an equity interest in Levitas, Inc., a company that develops biotechnology tools for cell sorting. UD’s interests were viewed and managed in accordance with the conflict of interest policies.
Acknowledgements This material is based upon work supported by Philips Healthcare. We thank the staff of the Stanford Healthy Aging study, particularly Thu Anh Vu, for their assistance regarding collection of discarded specimens.
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Highlights: (1) ToMMx platform has the advantages of being operated manually without dependence on a power-source especially establishing a unique fit for resource-limited settings. (2) ToMMx has the advantage of performing multiplexed detection of multiple cardiac biomarkers on-chip with high sensitivity, down to 5.29 pg/mL in patient samples. (3) The analytical results in a small cohort of 50 patient samples demonstrated that ToMMx platform can be applied to the detection of multiple cardiac biomarkers (cTnI, h-FABP, NTproBNP) with results compared side-by-side to commercial ELISA kits. (4) ToMMx is readily translatable given the ease of incorporating different biomarkers to detect. ToMMx is broadly applicable to other diseases and patient populations with welldefined biomarkers. The versatility of ToMMx allows the user to tailor the assay to a specific disease by generating a panel by utilizing target specific antibody pairs.
Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
UD is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions, (iii) Hillel Inc., a company focused on cell phone-based assays, and (iv) co-founder of and have an equity interest in Levitas, Inc., a company that develops biotechnology tools for cell sorting. UD’s interests were viewed and managed in accordance with the conflict of interest policies.
S0956-5663(19)31009-7
DOI:
https://doi.org/10.1016/j.bios.2019.111930
Reference:
BIOS 111930
To appear in:
Biosensors and Bioelectronics
Received Date: 24 July 2019 Revised Date:
12 November 2019
Accepted Date: 25 November 2019
Please cite this article as: Ozen, M.O., Sridhar, K., Ogut, M.G., Shanmugam, A., Avadhani, A.S., Kobayashi, Y., Wu, J.C., Haddad, F., Demirci, U., Total Microfluidic chip for multiplexed diagnostics (ToMMx), Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/j.bios.2019.111930. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
CRediT Author Statement Mehmet O. Ozen: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data Curation, Visualization, Writing – Original Draft. Kaushik Sridhar: Software, Formal Analysis, Writing – Review & Editing. Mehmet Giray Ogut: Visualization. Akash Shanmugam: Software, Resources. Anirudh S. Avadhani: Visualization. Yukari Kobayashi: Resources, Formal Analysis, Writing – Review & Editing. Joseph C. Wu: Supervision, Writing – Review & Editing. Francois Haddad: Resources, Formal Analysis, Writing – Review & Editing. Utkan Demirci: Conceptualization, Supervision, Writing – Review & Editing, Project Administration, Funding Acquisition.
Total Microfluidic chip for Multiplexed diagnostics (ToMMx) Mehmet O. Ozen a, b, Kaushik Sridhar a, Mehmet Giray Ogut a, Akash Shanmugam a, Anirudh S. Avadhani a, Yukari Kobayashi b, Joseph C. Wu b, c , Francois Haddad b, c and Utkan Demirci a, b * Affiliations: a
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early
Detection, Department of Radiology, School of Medicine Stanford University Palo Alto, CA 94304, US. b
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305,
US c
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305, US
* Corresponding author. Corresponding author’s e-mail: [email protected] (Utkan Demirci, Ph.D.)
Abstract Microfluidic technologies offer new platforms for biosensing in various clinical and point-of-care (POC) applications. Currently, at the clinical settings, the gold standard diagnostic platforms for multiplexed sensing are multi-step, time consuming, requiring expensive and bulky instruments with a constant need of electricity which makes them unsuitable for resource-limited or POC settings. These technologies are often limited by logistics, costly assays and regular maintenance.
Although there have been several attempts to miniaturize these diagnostic
platforms, they stand short of batch fabrication and they are dependent on complementary components such as syringe pumps. Here, we demonstrated the development and clinical testing of a disposable, multiplexed sensing device (ToMMx), which is a portable, high-throughput and user-friendly microfluidic platform. It was built with inexpensive plastic materials and operated manually without requiring electrical power and extensive training. We validated this platform in a small cohort of 50 clinical samples from patients with cardiovascular diseases and healthy controls. The platform is rapid and gives quantifiable results with high sensitivity, as low as 5.29 pg/mL, from only a small sample volume (4 µL). ToMMx platform was compared side-by-side with commercial ELISA kits where the total assay time is reduced 15-fold, from 5 hours to 20 minutes. This technology platform is broadly applicable to various diseases with well-known biomarkers in diagnostics and monitoring, especially with potential future impact at the POC settings.
Key words: Microfluidics, multiplexed sensing, on-chip manual biosensing, cardiovascular biomarkers
1. Introduction Blood tests are one of the standard tools used in clinic to diagnose various conditions and monitor diseases and the efficacy of therapeutic interventions (Gaggin and Januzzi, 2013; Ho et al., 2018; Huang et al., 2017; Mozaffarian et al., 2016; Omland and White, 2017; Vasan, 2006). The current biomarker detection systems; such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay or mass spectrometry, are accurate, well-established and sensitive assays. However, they require central laboratory settings and have limited potential for multiplexing. The cost is also a major limiting factor requiring significant human and material resources. Further, these assays are not suitable to satisfy the logistical and practical needs at point-of-care (POC) settings. POC devices have the advantage of sensing multiple biomarkers from a small volume of sample with shorter assay time compared to conventional assays (Verhertbruggen et al., 2009). To provide access to affordable POC monitoring, without trading off the ability to perform clinically relevant assays, we (Mani et al., 2016; Wang et al., 2014, 2011) and others (Adel Ahmed and Azzazy, 2013; Badu-Tawiah et al., 2015; Cheng et al., 2010; Fu et al., 2010; Mandon et al., 2010; Pollock et al., 2012; Sia et al., 2004; Sollier et al., 2009; Yu et al., 2013) have developed microfluidic biosensing platforms, based on specific capture and sensing of circulating biomarkers. However, none of these assays targeted multiple myocardial infarction and heart failure related biomarkers specifically and simultaneously. POC systems offer versatile, portable and easy-to-use platforms, which have lower production and analysis cost per sample, and eliminate requirements of trained personnel for application and interpretation of the resulting data (Drain et al., 2014). Developing a POC platform that may combine biomarkers from several mechanistic processes such as myocardial injury, wall stress, inflammation and oxidative stress in a simple cost-effective assay could improve care in resource scare areas.
Conventional POC diagnostic devices (e.g., lateral flow tests) have a wide-spread utility (Bruls et al., 2009; Drain et al., 2014), however, they generally can detect one or two biomarkers in series, and require compatible buffers and reagents for each target, where assays can crossreact and do not provide high sensitivity, precision, and quantitative results, making them unsuitable for cardiovascular biomarker detection (Carrio et al., 2015; Cho et al., 2009; Sher et al., 2017; Zhang et al., 2018). The advances in micro- and nano-technologies enable integration of POC diagnostic systems with well-established and sensitive quantitative assays (Krishnan et al., 2011; Tekin et al., 2013), such as ELISA, yielding miniaturized new POC platforms eliminating concerns with cross-reactivity (Bruls et al., 2009). However, widely used ELISA strategies need well-resourced clinical settings, and they are multi-step and can be labor intensive. Microfluidic technologies hold advantages over conventional platforms via lab-onchip applications to detect multiple biotargets from a small volume of biological samples at a relatively shorter assay time (Beyazkilic et al., 2016; Bruls et al., 2009; Dittmer et al., 2010; Laksanasopin et al., 2015; Ng et al., 2018, 2017; Piraino et al., 2016; Pollock et al., 2012; Sonawane et al., 2017; Song et al., 2012). The application of these principles to ELISA resulted in development of platforms for detection of various targets, including; cells, proteins, antibodies and lipids (Bruls et al., 2009; M. Cornaglia et al., 2014a, 2014b; Dittmer et al., 2010; Mani et al., 2016, 2011; Tekin et al., 2013; Wang et al., 2014; Woolley and Hayes, 2015). Even though there have been substantial proof-of-concept studies conducted, there is still limited number of studies performed for validation of cardiac biomarker sensing platforms, using actual clinical samples; and a translation-ready, pump- and power-free, clinical device for monitoring biomarkers in circulation has yet to be demonstrated.
Here, we developed a new platform for multiplexed detection of biomarkers from a small volume of plasma using rapid, portable, highly sensitive, quantitative, Total Microfluidics for Multiplexed diagnostics (ToMMx) platform. Then, we validated this platform by comparing it side-by-side with commercial ELISA kits evaluating clinical samples. This platform can be operated manually without sophisticated training, giving results within 20 min as the simplest and fastest manual ELISA on-a-chip ever reported for multiplexed biomarker detection. The platform is designed for simplicity so it can be produced using disposable and inexpensive plastic materials. To validate the system with clinical samples, we used cardiac troponin I (cTnI), heart type fatty acid binding protein (hFABP) and N-terminal pro B-type natriuretic peptide (NTproBNP), which are released from damaged and/or stressed cardiomyocytes, representing myocardial infarction and heart failure. These are chosen as model target molecules to validate the ToMMx platform (Gaggin and Januzzi, 2013; Omland and White, 2017). For optimal clinical utility, the platform has been optimized to have low and clinically relevant limit of detection (LOD) values, which were calculated as; 9.56 pg/mL, 92.5 pg/mL and 5.29 pg/mL for cTnI, hFABP and NT-proBNP, respectively in plasma. We used bio-banked samples collected from individuals
with
cardiovascular
diseases
(acute
coronary
syndrome
(ACS),
dilated
cardiomyopathy (DCM) and severe symptomatic artic stenosis patients undergoing transcatheter aortic valve replacement (AS)) and healthy controls. 2. Materials and Methods Reagents, materials, platform fabrication, characterization, optimization, sample processing in comparison with commercial assays and statistical analysis of the results can be found in the Supplementary Information.
3. Results and discussion 3.1. Design and chemistry of ToMMx platform We designed an on-chip ELISA technology, Total Microfluidic chip for Multiplexed diagnostics (ToMMx) platform, utilizing antibody functionalized magnetic beads as “mobile substrates”, prepared from polymethyl methacrylate (PMMA) and double-sided adhesive (DSA) polyethylene terephthalate (PET) film layers, all confined to a footprint of 7 cm x 8 cm (Fig. 1A and Fig. S1). ToMMx is laser-cut from these plastic sheets of different thicknesses and assembled using adhesive film layers between each layer (Fig. 1B). ToMMx platform chips are designed to have four lanes, three for biomarker detection and one internal control lane, which gives us ability to simultaneously evaluate the same sample for the presence of three different cardiovascular disease (CVD)-related biomarkers (Fig. 1B). Cardiac troponin-I (cTnI), hearttype fatty acid binding protein (hFABP) and N-terminal pro-brain natriuretic peptide (NTproBNP) are chosen as model CVD biomarkers to represent multiplexed biomarker detection capability of ToMMx. Static substrates, such as 96-well plate surfaces, have been used for performing traditional ELISA as a well-established method, where samples and different reagents are introduced into microwells sequentially according to the detection strategy desired (e.g., direct, indirect or sandwich ELISA) (Hosseini et al., 2018). We adopted similar assay steps and modified for ToMMx platform as; (1) magnetic bead functionalization with capture antibodies, (2) sample preparation, (3) reagent and sample loading on ToMMx, (4) analyte capture on magnetic beads, (5) biotinylated secondary antibody labeling, (6) streptavidin conjugated enzyme labeling, (7) TMB substrate catalysis and (8) colorimetric detection on spectrophotometer (Fig. 1C). These steps (4-7) are performed on the disposable ToMMx chip with magnetic beads that are actuated
from one chamber to another, housing assay reagents, washing buffer and mineral oil. To create an iterative filling into the chambers without mixing, we took advantage of surface tension differences between mineral oil versus water-based solutions. Reagents and buffers were preloaded on chip before sample processing, in an orderly fashion, where, first, we loaded oil chambers, then washing buffer chambers, and finally reagent chambers at the end (Fig. 1C, step 3). An assay stage to house the chip and a magnet holder to actuate beads during the assay through the assay route was designed and assembled using PMMA and DSA layers (Fig. S2).
Fig. 1. Schematic of the Total Microfluidic chip for Multiplexed diagnostics (ToMMx) platform and designed assay protocol. (A) Polymethyl methacrylate (PMMA) and double-sided adhesive (DSA) polyethylene terephthalate (PET) film layers of ToMMx design. (B) Laser-cut and assembled ToMMx. (C) Bead functionalization, sample preparation and assay steps of ToMMx. (1) Functionalization of tosylactivated magnetic beads with analyte specific primary antibodies. (2) Sample dilution buffer, plasma sample and functionalized beads mixed in tube as sample preparation. (3) Assay reagents, buffers and sample loading on ToMMx. (4) Analyte in the sample captured on antibody functionalized beads. (5) Analyte-antibody complex labeled with biotinylated secondary antibody. (6) Streptavidin conjugated poly-HRP binding to antibody-antigen-antibody sandwich complex. (7) TMB substrate catalysis by polyHRP in the complex. (8) Evaluation of analyte concentration via color change in the sample after transferring the colored liquid to a 96-well plate, mixing with stop solution and reading in with a spectrophotometer.
3.2. Simulating magnetic bead movement on ToMMx platform To better understand the bead-magnetic field interactions, in ToMMx, under the magnetic field applied by the neodymium magnets, we ran simulations (Fig. S3A-B). A three-dimensional (3D) model of ToMMx is used to calculate the magnetic flux density distribution of the magnets in various chambers of the device (Table S1). The magnetic susceptibility of PMMA, water-based buffers/reagents and oil used on ToMMx is much smaller compared to that of magnetic beads, hence, we neglect their contribution to magnetic field in the medium and approximated the magnetic field to be uniform. Simulations show that the magnetic forces increase as the distance between the beads and permanent magnets decreases (Fig. S3C). We compared three commercial magnetic beads of different sizes (1, 1.05, 2.8 µm in diameter, Table S2), and simulated the behavior of beads in terms of magnetic force, height and velocity over time in a chip chamber. Based on the multi-slice numerical simulation of the threedimensional model, the magnetic flux density in the positive z axis (Bz) is shown to vary from ~ 0.35 T (at the bottom of the chambers) to ~ 0.18 T (at the top of the chambers). Using this data, we estimate the time, velocity and force experienced by the beads on ToMMx. Magnetic beads with the highest magnetic susceptibility (
) respond better in chambers. According to
the simulations, the process takes 10 seconds for a magnetic bead to travel from the top of the chamber to the bottom. Then, we displace the magnets to a position under the consecutive compartment, where the beads can be efficiently transferred to the next chamber, following the magnet through the oil barrier, for the next assay step (Fig. S3D-F).
3.3. Analytical performance of the assay We first optimized the assay steps on a 96-well plate format by spiking target proteins in PBS, and established the limit of detection (LOD) values calculated as 0.4 pg/mL, 400 pg/mL, 25 pg/mL for cTnI, hFABP and NT-proBNP, respectively (Fig. 2A-C). To achieve a lower background signal for plasma samples, we evaluated four different sample diluents on the designed assay (Fig. S4). We chose two commercial diluents according to their higher signal-tonoise ratio for spiked plasma samples, and lower background signals for unspiked plasma samples (Fig. S5). We spiked plasma with target proteins and diluted with these diluents (cTnI and hFABP spiked plasma diluted with Plasma Diluent, NT-proBNP spiked plasma diluted with General Diluent) to define LOD on the 96-well plate format. We, then, calculated these LOD values as 2.5 pg/mL, 2.5 pg/mL, 40 pg/ mL for cTnI, hFABP and NT-proBNP, respectively (Fig. 2D-F).
Fig. 2. Assay optimization, limit of detection (LOD) in PBS and plasma. Target molecules (cTnI, hFABP and NT-proBNP) were spiked into PBS supplemented with 2% bovine serum albumin (BSA) and 0.05% Tween20. LOD values of the platform for three biomarkers were measured as 0.4 pg/mL (1.5%), 400 pg/mL (1.5%), 25 pg/ mL (6.6%) for (A) cTnI, (B) hFABP and (C) NT-proBNP, respectively. In a similar fashion, target molecules (cTnI, hFABP and NT-proBNP) were spiked into healthy plasma samples and diluted with optimized assay diluents. LOD of the platform for three biomarkers were observed as 2.5 pg/mL (5.9%), 2.5 pg/mL (2.7%), 40 pg/ mL (3.7%) for (D) cTnI, (E) hFABP and (F) NT-proBNP, respectively (). These LOD values were obtained on the 96-well format of the assay and the optimized parameters were translated on the ToMMx chip platform. Pink areas represent the average signal from six control samples (n=3, ± SD). Relative standard deviations (RSD) are given in brackets.
To reduce the background signal from plasma, we ran optimization steps for ToMMx platform (Fig. S5). We obtained high correlation (R2 ≥ 0.92) in comparison to the commercial ELISA kits when we evaluated the standard biomarkers (Fig. S6). Using the calibration curves obtained from these studies, we calculated Limit of Detection (LOD) and Limit of Quantitation (LOQ) of commercial kits and ToMMx platform (Table S3). LOD values are obtained as 9.56, 92.5, 5.29 pg/mL (10.1% RSD) for cTnI, hFABP and NT-proBNP, where LOQ values are found to be 28, 290 and 16.04 pg/mL, respectively for ToMMx. These LOD levels are significantly lower (around three orders of magnitude) compared to earlier reported on-chip ELISA strategies (Adel Ahmed and Azzazy, 2013). 3.4. Analytical performance of the platform in comparison with commercial kits We demonstrated analytical performance of cardiac biomarker biosensing on ToMMx, using clinical samples. We tested 38 patient samples (n = 11, acute coronary syndrome, ACS; n = 19, severe symptomatic aortic stenosis, AS; n = 8, dilated cardiomyopathy, DCM) and 12 control samples in head-to-head comparison with three commercial ELISA kits from two suppliers. Patient samples were obtained from the Healthy Aging study where patients were screened with London School of Hygiene dyspnea and chest pain questionnaire, echocardiograms and vascular ultrasound. Control samples were withdrawn from healthy individuals without evidence of clinical or subclinical cardiovascular disease. In addition, these individuals did not have a history of inflammatory conditions, cancer or acute infection. As shown in Table S4, patients with AS were older compared to remaining individuals. Left ventricular systolic function was preserved in patients with ACS, while it was reduced in patients with AS, and remarkable reduced in individuals with DCM. In ACS, cTnI levels tend to increase due to myocyte cell damage, where cytosolic hFABP leaks to circulation becoming detectable as well (Bjurman et al., 2015; Kurz et
al., 2011; Lippi et al., 2013; Schulz et al., 2007; Zhou et al., 2014). NT-proBNP is being monitored to diagnose and prognose individuals with heart failure, since circulating NT-proBNP concentration increases due to the increased left ventricular load (Januzzi et al., 2006, 2005; Ozturk et al., 2011; Weber and Hamm, 2006; Yancy et al., 2013; Yeo et al., 2003). Using ToMMx, we were able to distinguish control samples from individuals, who had one of the following conditions; ACS, AS and/or DCM, significantly with high sensitivity (Fig. 3A-F). When we computed the obtained data to prepare the receiver operating curves (ROC) for each biomarker, the area under the curve (AUC) values were depicted as 0.904, 0.741, and 0.750 for cTnI, hFABP and NT-proBNP, respectively on ToMMx (Fig. 3G-I). We also analyzed the results separating the samples as “diseased” and “control” for the presence of these three biomarkers. The signals obtained from diseased samples had statistically higher signal when ToMMx was used compared to commercial kits (Fig. S7A-C). The signals obtained from control samples were not significantly different between two methods (Fig. S7D-F). We, also, analyzed these results using Bland-Altman method, where 3 patient samples for cTnI, 2 patient samples for hFABP and 3 patient samples for NT-proBNP, and 1 healthy sample for all biomarkers exceeded the 95% confidence level (±1.96 SD) (Fig. S8). Furthermore, we evaluated looked for the presence of; cTnI and hFABP in 11 ACS patients, NT-proBNP in 19 AS and 8 DCM patients. Among these tested samples, ToMMx resulted with higher signals for cTnI, hFABP and NT-proBNP (for AS) detection in comparison to commercial kits (Fig. 4A-C). For NT-proBNP on DCM patients, both ToMMx and commercial kits gave comparable results (Fig. 4D). When ToMMx was used, we reached to ~ 91% accuracy to identify ACS (via cTnI and hFABP), and ~95% accuracy to identify AS (via NT-proBNP). We were able to also identify all DCM patients using NT-proBNP as a diagnostic biomarker (Fig. 4E-H).
Fig. 3. ToMMx vs commercial ELISA kits. We evaluated the presence of cTnI, hFABP and NTproBNP in 38 patient samples in comparison to 12 healthy plasma samples using (A-C) ToMMx platform and (D-F) commercial ELISA kits. Mann-Whitney unpaired t-test used to analyze the results. ToMMx platform was able to distinguish patient samples from healthy samples, significantly. Results obtained from commercial ELISA kits were non-significant. ROC curves present AUC values for (G) cTnI, (H) hFABP, (I) NT-proBNP (n=3-4, ± SD).
Fig. 4. Disease specificity. We analyzed the patient plasma results obtained from ToMMx via cTnI and hFABP for MI (n = 11), and via NT-proBNP for TAVR (n = 19) and DCM (n = 8) identification. ToMMx significantly differentiated, (A) cTnI (p = 0.038) and (B) hFABP (p=0.033) presence for MI, and (C) NT-proBNP (p=0.0022) for TAVR detection, where (D) NTproBNP results for DCM were not significantly different compared to commercial ELISA kit results. Mann-Whitney paired t-test used to analyze the results. Bland-Altman analysis performed to analyze the difference between methods for (E) ACS – cTnI, (F) ACS – hFABP, (G) AS - NT-proBNP, and (H) DCM - NT-proBNP. The 95% confidence intervals on the mean is shown on the graphs with red-dotted lines as ± 1.96 SD (n = 3-4, ± SD).
Cardiac diseases are major adverse events, where the interaction of tissues and cells in the cardiac niche, necessitate multiple biomarker approaches to decode their interplay through disease progression. The complexity within this network makes CVDs complex disorders, which can be potentially better identified, exercising multiple biomarker monitoring approaches, rather than focusing on a single biomarker (Bayes-Genis and Ordonez-Llanos, 2015; Dhingra and Vasan, 2017; Doehner, 2012; Giannessi, 2011; Ho et al., 2018; Vasan, 2006; Wang et al., 2017). Standard cardiac biomarker testing in routine practice consists of blood collection, plasma or serum separation and testing samples either using an automated platform in a centralized laboratory or using a commercial ELISA kit. These approaches are expensive and time consuming for daily practices, requiring trained personnel and routine device maintenance. Some of these, over time, become inaccurate due to delayed maintenance of the large-scale equipment associated with high maintenance costs, given that many of these centralized labs are not wellfunded, especially in developing regions. Moreover, especially in resource-limited settings, centralized sample processing and evaluation can bring complications such as lost samples and results. Further, the need for a centralized laboratory can significantly deaccelerate and delay acquisition and dissemination of time sensitive clinical results, where the POC diagnostic tests are available. To eliminate dependence on electrical power on sample processing, we optimized a system for manual operation considering resource-limited setting where the electrical power shut-downs are common and hence, we did not intend to take an automation path with the system. The developed platform has a lower sample analysis cost and there is no ELISA-based portable platform for POC that we can compare our test against, that is commercially available from a cost perspective. However, manufacturing cost of the chip relies on multiple variables,
including the ordered components, their production methods and distance to manufacturing site, as well as transportation costs. We anticipate that our device can be ultimately produced at a low cost given that it can be batch-fabricated by injection molding. The cost of the plastic pieces of ToMMx, PMMA and DSA layers, can be produced at a cost as low as 10 cents, and buffers of the assay can be purchased for 10 cents. Antibodies and magnetic beads constitute the remaining ~ 95% of the total cost that can be potentially lowered by bulk orders for a commercial setting, at production scale workflows. Earlier magnetic bead-based ELISA systems have been reported by our lab and others (Adel Ahmed and Azzazy, 2013; Mani et al., 2016; Wang et al., 2014) however these systems did not present a multiplexed CVD biomarker sensing capability. Our approach presents a comprehensive study done in obtaining magnetic force of the magnetic beads to precisely estimate the time and velocity that should be followed to reach the best time and magnet movement in each chamber to achieve the optimum workflow, and hence, enhancing the assay performance. Minimizing / Eliminating sample preparation steps is one of the significant advantages of microfluidic platforms. Several strategies, that are either already commercialized or in the pipeline, utilizing fluid dynamics (Robinson et al., 2017), filtration (Tasso, n.d.), or micro needles (Blicharz et al., 2018), can be adapted to ToMMx platform easing the path for POC applications. This study focuses on the capability of an on-chip platform to perform multiplexed analysis resulting with clinically relevant LODs at minimal necessities. Current version of ToMMx is used in a laboratory environment evaluating plasma samples with the help of a spectrophotometer as a result enumeration strategy as a proof-of-concept study. With the further studies on buffers, bead concentrations and portable reading strategies ToMMx will be fully
adaptable for POC settings, minimizing assay steps such as using a stop solution, which we implemented in here to reduce the effect of sample transfer on the results. With further development and integration portable readers, this system has great potential for POC applications that would make the whole system more portable and easier to use eliminating the transfer steps and replacing the stop reactions with timed measurements (Inci et al., 2015; Liang et al., 2017; Wang et al., 2011). 4. Conclusion In this study, we designed a novel platform that combines the advantages of sensitivity, multiplexing, minimal sample volume, simplicity, low-cost and time effectiveness to sense multiple cardiac biomarkers simultaneously from a small volume of plasma sample. We validated the platform for three key cardiovascular biomarkers reflecting the pathway of myocardial injury (cTnI, h-FABP) and myocardial wall stress (NT-proBNP) in a small cohort of clinical samples. The whole assay can be performed manually by actuating magnetic beads with a simple off-the-shelf magnet without the requirement of a complex instrument. The quantitative results are precise and accurate compared to plate ELISA systems. Also, the total assay time is reduced 15-fold, with the help of reduced sample volume, increased assay surface area on magnetic beads, higher surface-to-volume ratio, and step-by-step compartmentalization of the assay on-chip. Developed platform performed well in sensitivity and specificity compared to commercial ELISA kits with significantly lower LOD values. This feature of our platform enabled us to detect targets within the clinically relevant cut-off concentrations (9.56 pg/mL for cTnI, and 5.29 pg/mL for NT-proBNP) only using a minute amount of a sample. Studies presented in this work establish the validation and clinical relevance, to further translate the tool into field studies.
In conclusion, we anticipate that our device will ultimately represent a laboratory-quality, multiplexed platform, utilizing the advantages of microfluidics to build a portable, highthroughput and user-friendly tool that can be performed by any individual with minimal training, simplifying and reducing the cost of diagnosis and monitoring of cardiovascular events. Conceivably, the development of this versatile, multiplex platform, with its flexible design, could facilitate the emergence of other clinically relevant POC assays with well-defined clinically important markers at multiple settings; including resource-limited settings, home, primary care, and bedside at the hospitals, after integration of ToMMx with portable readers, such as mobile phones.
CRediT authorship contribution statement Mehmet O. Ozen: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data Curation, Visualization, Writing – Original Draft.
Kaushik Sridhar:
Software, Formal Analysis, Writing – Review & Editing. Mehmet Giray Ogut: Visualization. Akash Shanmugam: Software, Resources. Anirudh S. Avadhani: Visualization. Yukari Kobayashi: Resources, Formal Analysis, Writing – Review & Editing. Francois Haddad: Resources, Formal Analysis, Writing – Review & Editing. Joseph C. Wu: Supervision, Writing – Review & Editing. Utkan Demirci: Conceptualization, Supervision, Writing – Review & Editing, Project Administration, Funding Acquisition.
Declaration of competing interest UD is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions, (iii) Hillel Inc., a company focused on cell phone-based assays. (iv) co-founder of and have an equity interest in Levitas, Inc., a company that develops biotechnology tools for cell sorting. UD’s interests were viewed and managed in accordance with the conflict of interest policies.
Acknowledgements This material is based upon work supported by Philips Healthcare. We thank the staff of the Stanford Healthy Aging study, particularly Thu Anh Vu, for their assistance regarding collection of discarded specimens.
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Highlights: (1) ToMMx platform has the advantages of being operated manually without dependence on a power-source especially establishing a unique fit for resource-limited settings. (2) ToMMx has the advantage of performing multiplexed detection of multiple cardiac biomarkers on-chip with high sensitivity, down to 5.29 pg/mL in patient samples. (3) The analytical results in a small cohort of 50 patient samples demonstrated that ToMMx platform can be applied to the detection of multiple cardiac biomarkers (cTnI, h-FABP, NTproBNP) with results compared side-by-side to commercial ELISA kits. (4) ToMMx is readily translatable given the ease of incorporating different biomarkers to detect. ToMMx is broadly applicable to other diseases and patient populations with welldefined biomarkers. The versatility of ToMMx allows the user to tailor the assay to a specific disease by generating a panel by utilizing target specific antibody pairs.
Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
UD is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions, (iii) Hillel Inc., a company focused on cell phone-based assays, and (iv) co-founder of and have an equity interest in Levitas, Inc., a company that develops biotechnology tools for cell sorting. UD’s interests were viewed and managed in accordance with the conflict of interest policies.