Risk stratification of heart failure from one drop of blood using hand-held biosensor for BNP detection
Introduction
Molecular biomarkers that provide physio-pathological information play an important role in the diagnosis and prognosis of diseases such as cardiovascular diseases (CVDs) and cancers (Mayeux, 2004, Liu et al., 2014). Timely diagnosis aids in the prevention of CVDs, which are the leading causes of death worldwide. Continuous monitoring of molecular biomarkers provides additional risk stratification for CVDs, beyond the traditional biomarkers (Gilstrap and Wang, 2012, Yin et al., 2014). High rates of morbidity and mortality could be prevented through regular assessment and management of cardiovascular risk, by providing cost-effective and convenient means of health monitoring systems available to all. Congestive heart failure (CHF) is essentially the inability of the heart to pump blood to all parts of the body, due to weakened heart muscles or defects (Taylor, 1996). Since heart failure can be presented with a variety of symptoms, physicians face challenges in definitive diagnosis and correct prognosis. Protein based biomarkers of heart failure such as brain natriuretic peptide (BNP) and N-terminal pro brain natriuretic peptide (NT-proBNP) are identified as the standard biomarkers for diagnosis and prognosis of heart failure (Lin et al., 2014, Natriuretic Peptides Studies, 2016). Clinical determination of the natriuretic peptides from blood plasma can be performed using conventional spectroscopic techniques such as radioimmunoassay and chemiluminescence (Cowie et al., 2003, Nishikimi et al., 2013). More recently surface plasmon resonance (SPR) based immunoassays and electrochemical enzyme immunoassays have also been developed to quantify BNP levels (Matsuura et al., 2005, Jang et al., 2014). However, these assays are quite complex, performed using bulky and costly equipment, often requiring trained laboratory staff to perform the assay.
Miniaturized electronic sensors present an opportunity to perform high sensitivity immunoassays at considerably low cost. The ease of integration of these miniaturized biosensors with the measurement system facilitates compact and portable sensor units that can be used for point-of-care assays. In this genre, field effect transistor (FET) based biosensors are highly desirable owing to its characteristic features such as high sensitivity, low cost, easy signal read-out and fast response times. However, FET based sensors suffer from charge screening effect in high ionic strength solutions such as blood due to the extremely short Debye length (Stern et al., 2007, Zheng and Lieber, 2011). As a result, extensive sample pre-treatment methods are adopted to facilitate biomolecule detection, such as dilution and desalting (Zheng et al., 2005, Zheng and Lieber, 2011, Stern et al., 2010). This severely impacts the reliability and practicality of FET based assays in clinical applications especially if a rapid, inexpensive in-vitro diagnostic platform is desired.
In the present research, we have devised and characterized a hand-held biosensor system for the determination of BNP levels from a single drop of whole blood. The sensing methodology is based on extended gate electrical double layer (EDL) FET biosensor that can directly detect the target analyte in high ionic strength solution such as whole blood, offering bio sensing beyond the Debye length. EDL FET biosensors have demonstrated high sensitivity and selectivity in biomolecule detection (Chu et al., 2017). In the extended gate design, device to device variations are considerably reduced allowing easy sensor calibration for variety of clinical applications. Also, FET is protected from harsh chemical environments of the physiological fluids, providing enhanced stability and robustness. The use of single FET for multiple electrical measurements significantly lowers the assay cost. In here, we demonstrate the detection of BNP in 1 × PBS with 4% BSA and clinical whole blood samples obtained from different patients. The sensor exhibits very high sensitivity, selectivity and wide dynamic range of detection. Simple gravitational separation of blood cells from blood plasma is performed, without any external actuation to eliminate any interference of cell components of whole blood. A hand-held biosensor measurement unit is developed to integrate the sensor chip and the electrical test results are displayed in the LCD display. We demonstrate that our unique sensing methodology can facilitate rapid screening of protein biomarkers from a single drop of whole blood (< 10 µL) in 5 min, and the simplistic assay can be performed by consumers with minimal protocols, as per their convenience and requirements.
Section snippets
Fabrication of sensor array chip
A simple sensor array chip is fabricated on an epoxy substrate, containing multiple gold electrode pairs. Epoxy substrate is fabricated by pouring thermo-curable epoxy resin in PDMS mold and curing at temperatures of 125 and 165 ℃ for 1 and 1.5 h respectively. Post curing, electron beam (e-beam) evaporator technique is used to deposit gold electrode pairs (Ti (200 Å) and Au (2000 Å)) on the epoxy substrate. Typically, an eight-electrode pair array is fabricated. The two electrodes forming the
Results and discussion
The schematic representation of the extended gate design of EDL gated FET biosensor is shown in Fig. 1(a). The real image of the sensor mounted on the handheld biosensor system is shown in Fig. 1(b). The sensor structure is uniquely different from traditional FET based biosensors. In the extended gate design, the gate metal is connected to a pair of gold electrodes which are separated by a short gap. The gold electrode pair simulate a two-plate capacitor, with the test solution (containing the
Conclusion
In this research, we have devised a hand-held diagnostic system based on extended gate EDL FET biosensor for the detection of BNP as a biomarker for congestive heart failure. We have developed FET sensor methodology to directly detect protein biomarkers in whole blood, without extensive sample pre-treatments. This methodology is based on the high field generated across the test solution which modulates the FET channel conductivity, enabling detection beyond Debye length in high ionic strength
Acknowledgement
This work was partially supported by research grants from Ministry of Science & Technology (MOST 106–2221-E-007-002), (MOST 106–2218-E-007–015-MY2), NTHU-Hsinchu Mackay Memorial Hospital 2017 joint project, National Yang-Ming University (2017 SPARK) and National Tsing Hua University (106N523CE1). We thank the technical support from National Nano Device Laboratories (NDL) in Hsinchu and the Center for Nanotechnology, Materials science, and Microsystems (CNMM) at National Tsing Hua University.
References (25)
- et al.
Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing
Biophys. J.
(1995) Biomarkers: potential uses and limitations
NeuroRx
(2004)- et al.
Enumeration of circulating tumor cells and investigation of cellular responses using aptamer-immobilized AlGaN/GaN high electron mobility transistor sensor array
Sens. Actuators B: Chem.
(2018) - et al.
Charge displacement by adhesion and spreading of a cell
Bioelectrochemistry
(2001) - et al.
The extended gate chemically sensitive field effect transistor as multi-species microprobe
Sens. Actuators
(1983) - et al.
Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum
Sci. Rep.
(2017) - et al.
Clinical applications of B-type natriuretic peptide (BNP) testing
Eur. Heart J.
(2003) - et al.
Micromotion of mammalian cells measured electrically
Proc. Natl. Acad. Sci. USA
(1991) - et al.
Biomarkers and cardiovascular risk assessment for primary prevention: an update
Clin. Chem.
(2012) - et al.
Ultrasensitive and ultrawide range detection of a cardiac biomarker on a surface plasmon resonance platform
Anal. Chem.
(2014)