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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.

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© 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: utkan@stanford.edu (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 cross-

react 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-on-

chip 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 (NT-

proBNP), 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), heart-

type fatty acid binding protein (hFABP) and N-terminal pro-brain natriuretic peptide (NT-

proBNP) 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 pre-

loaded 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 tosyl-

activated 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 poly-

HRP 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 (3-

D) 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 three-

dimensional 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-to-

noise 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 NT-

proBNP 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) NT-

proBNP 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 well-

funded, 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, high-

throughput 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, NT-

proBNP) 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 well-

defined 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.