Elsevier

Biosensors and Bioelectronics

Volume 128, 1 March 2019, Pages 176-185
Biosensors and Bioelectronics

Paper microfluidic device for early diagnosis and prognosis of acute myocardial infarction via quantitative multiplex cardiac biomarker detection

https://doi.org/10.1016/j.bios.2018.12.049Get rights and content

Highlights

  • The first POC device developed for early diagnosis and prognosis of AMI.

  • Wax printed µPAD allows rapid multiplex detection of GPBB, CK-MB and cTnT.

  • Developed µPAD has potential use in clinical testing.

  • Results obtained are well correlated with Siemens Centaur XPT Immunoassay system.

Abstract

The early detection of acute myocardial infarction (AMI) upon the onset of chest pain symptoms is crucial for patient survival. However, this detection is challenging, particularly without a persistent elevation of ST-segment reflected in an electrocardiogram or in blood tests. A majority of the available point-of-care testing devices allow accurate and rapid diagnosis of AMI. However, AMI diagnosis is reliable only at intermediate and later stages, with myocardial injury (> 6 h) and MI, based on the expression of specific cardiac biomarkers including troponin I or T (cTnI or cTnT), creatine kinase-MB (CK−MB), and myoglobin. Diagnosis at the early myocardial ischemia stage is not possible. To overcome this limitation, a sensitive and rapid microfluidic paper-based device (µPAD) was developed for the simultaneous detection of multiple cardiac biomarkers for the early and late diagnosis of AMI. The glycogen phosphorylase isoenzyme BB (GPBB) was detected during early (within first 4 h) ischemic myocardial injury. On the same µPAD platform, detection of prolonged elevation of levels of cTnT and CK-MB, which are only produced 6 h after the onset of chest pain in human serum, was possible. Sandwich immunoassay performed on the µPAD achieved reproducibility (RSD approximately 10% and intra-and inter-day precision (CV 10–20%, 99th percentile), as well as consistently stable test results for 28 days, with strong correlation (r2 = 0.962), using the standard Siemens Centaur XPT Immunoassay system. The present findings indicate the potential of the µPAD platform as a point-of-care device for the early diagnosis and prognosis of AMI.

Introduction

In the clinical setting, the term acute coronary syndrome (ACS) refers to a spectrum of clinical presentation for symptoms of ischemia (inadequate blood supply to heart), such as unstable angina, non-ST elevation myocardial infarction (STEMI), and STEMI (Overbaugh, 2009). Chest pain is one of the most common complaints among patients presenting to the emergency department. The diagnosis of ACS is dependent on evidence of myocardial ischemia on an electrocardiogram (ECG) and evidence of myocardial injury by determining the levels of cardiac biochemical markers (Ryu et al., 2011). For patients with chest pain, no evidence of myocardial injury based on the levels of cardiac biomarkers in the blood is considered to present unstable angina, whereas patients presenting with positive cardiac biomarkers, with or without electrocardiographic ST-segment depression or T wave inversion, are undergoing non-STEMI due to a relatively small damage of heart muscles (Anderson et al., 2007, Bertrand et al., 2000). Furthermore, patients with ST-segment elevation on an ECG due to high damage extent of heart muscles indicate acute STEMI (Van de Werf et al., 2003) (Supplementary material Fig. S1).

In clinical trials, patients with chest pain and suspected ACS may be referred urgently to the emergency department and undergo ECG monitoring to aid in risk stratification (Lang et al., 2000). However, misinterpretation of findings on ECG accounts for 23–40% of misdiagnosed MI cases (Kontos et al., 2010). Due to ST-elevation, MI can be readily diagnosed with culprit ECG findings. However, for non-ST elevation, MI diagnoses are more challenging. Therefore, serial cardiac biomarker sampling is crucial for the diagnosis of acute MI in patients with non-diagnostic ECGs or chest discomfort symptom. Serial measurement of cardiac biomarkers of myocardial injury, such as cardiac troponin (T or I), creatine kinase-MB (CK-MB), and myoglobin, are widely accepted as important determinants in ruling out MI in the emergency department (Al-Hadi and Fox, 2009, De Winter et al., 1995).

Currently available routine biomarkers of myocardial injury exhibit inadequate sensitivity for determining early MI, and none of these can be used to detect myocardial ischemia. Cardiac troponin (T or I) and CK-MB are only detectable for myocardial injury after 6 h of chest pain onset, and repeated measurement is necessary at 8–12 h after admission if a negative result is obtained at initial presentation (< 6 h) (Bertrand et al., 2000). Moreover, the earliest marker, myoglobin, which is detectable within the first 3 h of chest pain onset, is reportedly more specific for skeletal muscle injury than that for myocardial injury (De Winter et al., 1995, Mair et al., 1992). Hence, early diagnosis of acute chest pain by blood testing is still difficult in patients without persistent ST-segment elevation as most of these patients have been diagnosed as having unstable angina because of myocardial ischemia during initial presentation, with no elevation of cardiac marker levels (Mythili and Malathi, 2015). Early diagnosis of ischemic myocardial injury is essential for effective management of patients suspected with ACS, thereby avoiding life-threatening situations, such as heart attack (MI).

To overcome these limitations, glycogen phosphorylase isoenzyme BB (GPBB), which can diagnose myocardial injury and ischemia, was introduced as a reliable early marker to replace myoglobin for detection of myocardial injury (Bozkurt et al., 2011, Cubranic et al., 2012). GPBB activation in myocardial ischemia occurs because of an increase in glycogen degradation, which releases GPBB in the blood circulation within the first 4 h after chest pain onset before myoglobin, CK-MB, and troponins.

With the aim of miniaturizing the immunoassay procedures presently done at the central laboratory to a portable device that can be more widely used, and to reduce the waiting time for emergency room patients, lateral flow assays have been developed and are commercially available as point-of-care testing (POCT) devices from a variety of manufactures for cardiac biomarker measurement in single and/or multiplex strategy for qualitative and/or quantitative detection with a portable reader (Amundson and Apple, 2015, McDonnell et al., 2009). All the troponin (T or I), CK-MB, and myoglobin analytes can be read qualitatively based on the color change and quantified individually and/or simultaneously with a reader within approximately 10–20 min after addition of the sample to the POCT device. Other methods studied more recently include fluorescence (Cai et al., 2018, Cho et al., 2014, Kim et al., 2014, Lee et al., 2013), magnetic beads (Ryu et al., 2011), and surface enhance Raman spectroscopy (SERS) tags (Zhang et al., 2018a) to enhance the detection sensitivity at lower concentrations and improve the wider dynamic range to cover the clinical symptoms. However, a dedicated reader is required for quantification of fluorescence or Raman signal (Table 1).

Moreover, lateral flow assays have some limitations in terms of flexibility in platform design for multianalyte detection and detection sensitivity for multi-analytes on a single test-strip. Owing to space limitation on a lateral flow strip, additional test lines are always drawn over a piece of membrane or few strips are overlapped together at the sides of the sample pad for multiplex analyte detection This attempt does not increase the risk of false binding, since each assay is performed in parallel and independently, which removes the possibility of cross-contamination. The main drawback of this approach, however, is the amount of sample required, which increases as a function of the number of strips added (and consequently of biomarkers to be measured). Thus, it is difficult to construct a flexible design on a lateral flow test strip, such as including several reaction zones on the membrane. Additionally, the detection sensitivity for multiplex assay on a single test strip may be reduced because of the use of large amounts of capturing and labeled detecting antibodies, which may increase the risk of false positivity due to non-specific binding with non-target analytes. However, this limitation depends on whether the labeled antibodies are embedded in the lateral flow device and encounter all the capturing antibodies during their flow. This problem has been not solved. Thus, the lateral flow assay limits the number and types of biomarkers that can be detected in a multiplex assay. Several research studies have been reported where smartphone-based test strip readers based on color analysis were used for various applications (Lopez-Ruiz et al., 2014, Oncescu et al., 2014, Oncescu et al., 2013, Shen et al., 2012). However, the commercial lateral flow assays that are currently available usually require a proper readout device for highly sensitive and quantitative detection during clinical diagnostic testing.

Herein, the microfluidic paper-based device (µPAD) was tested and demonstrated to exhibit high multiplexing capability and flexibility in the assay design. This design could be easily adapted to paper by various fabrication designs (Dincer et al., 2017). Moreover, the detection sensitivity of the multiplex assay on the fabricated µPAD could be further enhanced by utilizing numerous antibody conjugates for specific multiplex analyte detection. Nanoparticles (NPs) are preferably used as labels for colorimetric detection using µPAD. They offer distinct advantages. They allow multiplexed analysis by using differently colored NPs that are visible without an external excitation source (compared to fluorescence and quantum dot label) and are resistant to photobleaching. For instance, gold nanoparticles (AuNPs) (Choi et al., 2010, Quesada-Gonzalez and Merkoci, 2015, Rong-Hwa et al., 2010, Veigas et al., 2012) and silver nanoparticles (AgNPs) (Yen et al., 2015) can display different colors within the visible spectrum tuned by variations in shape and size that allow easy visual distinction. In addition, NPs also have high binding efficiencies with a relatively low usage of antibody during bioconjugation due to the larger surface area to volume ratio (Arruebo et al., 2009).

To overcome the requirement of an expensive analyzer and/or reader for quantification, a reflectance detector, such as a mobile phone camera and/or scanner, was presently explored as an alternative low-cost and portable readout device that could be used with the µPAD for colorimetric detection. The intensity of color generated in a spatially defined zone on µPAD is converted to a number of pixels based on the RGB (red/green/blue) color analysis to generate a calibration plot for any quantitative analyte measurement (Martinez et al., 2010, Sechi et al., 2013).

In the present study, we aimed to develop a µPAD for multiple marker detection for diagnosis of AMI using early (GPBB) and late (CK-MB and cTnT) cardiac biomarkers (Supplementary material Fig. S2). Considering the fact that none of the single markers has exhibited sufficient diagnosis accuracy for acute myocardial infarction, a novel combination of these three specific cardiac biomarkers, viz., GPBB for early diagnosis (within the first 4 h) and cTnT and CK-MB for late after symptom onset (> 6 h) and prognosis of AMI (7–14 days), was incorporated into one device (Scheme 1). This enables the identification of multiplex biomarker release profiles for improved sensitivity and can identify MI (STEMI) and at-risk patients (non-STEMI and unstable angina) with a possible life-threatening cardiac event. The µPAD was fabricated by a simple and rapid fabrication technique (approximately 5 min) involving wax printing on a paper substrate in the form of a nitrocellulose (NC) membrane card using a solid wax printer (Fuji Xerox ColorQube 8870) to permit multiplexing branched flow into separate test zone(s) by capillary action for simultaneous multiplex detection (Supplementary material Fig. S3). The protocol was slightly modified from that of Carrilho et al. (2009a). To enhance the specificity, sensitivity, and visual judgment of the multiplexing assay, the nanomaterial used as optical labels (AuNPs, AgNPs, and gold urchin NPs) were separately conjugated with their specific detecting antibody to provide a distinct visible color as the colorimetric signal, e.g., AuNPs–cTnT, red; AgNPs–GPBB, yellow; and gold urchin NPs–CK-MB, purple. The color labels were assessed using a camera (with imaging software) to quantitatively measure the color intensity produced at the test zone(s). This is because the light reflected from each surface of the test zone(s) can be detected easily with the available reflectance detectors, such as a desktop scanner, digital camera, or phone camera. Thus, the NPs utilized by the µPAD as labels are robust and simple, and allow qualitative, semi-quantitative, and quantitative determination of the biomarkers.

Section snippets

Materials and methods

Please refer to Supplementary material for Research Methodology.

Qualitative detection of multiplex cardiac biomarkers on the fabricated µPAD

Qualitative analysis in healthcare has become increasingly important to provide immediate results for the direct detection of the presence or absence of biomarkers in the sample to diagnose patients suspected of AMI. Qualitative test results can aid clinicians in decision-making at a critical moment for admitting a patient with MI. The incorporation of metal nanoparticles, viz., gold, silver, and gold urchin, as labels allows visual examination of the color signal at the reaction zone(s),

Conclusions

An inexpensive and disposable µPAD was developed for simultaneous colorimetric determination of multiplex cardiac markers by using only an available phone camera and/or desktop scanner for quantification, which significantly decreased the analysis time (10 min) and reduced the cost for multiple marker measurement. A combination testing of the early marker GPBB with CK-MB and cTnT on the µPAD offers the best approach to include all symptoms of ACS for early diagnosis (ischemic myocardial injury)

Acknowledgements

This work was financially supported by the University of Malaya postgraduate grant (PPP) (PG021–2015A), the Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education of Malaysia (MOHE) (FP041–2016), and the Research University Grant (GPF057B-2018).

Credit author statement

Wei Yin Lim carried out the experiments and wrote the manuscript. Sook Mei Khor and Boon Tong Goh supervised the project. Sook Mei Khor conceived the original idea, provided financial support, reviewed and edited the manuscript. Boon Tong Goh contributed in fabrication of microfluidic-paper based device, provided financial support and resources for the project and revised the manuscript. T. Malathi Thevarajah provided resources and contributed in the validation of human serum analysis in this

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