The development of electrochemical biosensors for cholesterol

Coronary vascular disease (CVD) is the number one cause of death worldwide. According to a WHO report and global and regional projections of mortality and burden of disease, by 2030, the number of people dying from heart disease and stroke will increase to reach 23.3 million. Non-HDL cholesterol, determined by subtracting the high density lipoprotein cholesterol (HDL-C) concentration from the total cholesterol (TC) content, has been recommended as a target for preliminary CVD prevention. In recent times, single-step homogeneous assays have been developed which allow simple and selective measurement of cholesterol fractions. Electrochemical sensors have also been developed which are based on the electrocatalysis of hydrogen peroxide using low cost printed sensor methodologies and such platforms would have the potential to be used as the basis of fabricating cholesterol biosensors for point of care use.Here, the development of electrochemical biosensors for the selective measurement of HDL-C, TC, and by subtraction non-HDL-C was explored. A spectrophotometric assay for use at room temperature and with minimal sample dilution was first established in order to optimise the assay reagent components for the development of selective cholesterol assays. Assay chemistries based on polyoxyethylene tribenzylphenyl ethers (Emulgen B-66) and Triton X-100 for the selective measurement of HDL-C and TC, respectively, were developed. The impact of these reagents on the electrocatalytic reduction of hydrogen peroxide at silver paste screen printed electrodes was also evaluated and optimised.Electrochemical biosensors for HDL-C and TC using externally added assay reagents were developed by combining the homogeneous assay methodologies with the printed electrocatalytic electrodes. The effects of assay reagents such as surfactants, enzymes, HDL-C sample and delipidated serum on the electrode behaviour were assessed amperometrically in the presence of hydrogen peroxide solutions. The electrodes showed increases in their catalytic activity toward hydrogen peroxide in the presence of both selective and non-selective surfactant and decreases in the presence of cholesterol oxidase and HDL-C samples. Despite the negative effects of cholesterol oxidase and sample matrix on electrode behaviour, the electrode response was linear within the clinically relevant ranges of HDL-C and TC. The modified electrodes were evaluated for their ability to selectively measure HDL-C and TC in clinical serum samples. The resulting HDL-C biosensor yielded a sensitivity of 3.32×10-8 A/mM with a linear range of 0 to 4 mM (r2=0.999), LOD of 0.5 mM and average RSD of 9.5% (n=5) while the TC biosensor had a sensitivity of 2.24×10-8 A/mM and a linear range of 0 to 10 mM r2=0.984), LOD of 2 mM and average RSD of 10.8% (n=3). The correlation between the HDL-C sensor and a commercial laboratory assay in clinical serum samples had a slope of 0.87 and a Pearson correlation coefficient of 0.76 (n=13) while the correlation for TC measurement had a slope of 1.07 and a Pearson correlation coefficient of 0.87.Finally, in order to develop a disposable biosensor suitable for point of care testing, integrated biosensors for HDL-C and TC were fabricated by inkjet-print deposition of assay reagents on the electrode surface. Integrated biosensors for the measurement of HDL-C were optimised and yielded a sensitivity of 4.55×10-8 A/mM with a linear range of 0 to 4 mM (r2=0.993) with an LOD of 0.25 mM and average RSD of 6.6% (n=3). The integrated TC biosensor had a sensitivity of 9.38×10-9 A/mM and linear range of 0 to 9 mM (r2=0.982), LOD of 0.5 mM and average RSD of 9.5% (n=3)

Biomedical Diagnostics Enabled by Integrated Organic and Printed Electronics

© 2017 American Chemical Society. Organic and printed electronics integration has the potential to revolutionize many technologies, including biomedical diagnostics. This work demonstrates the successful integration of multiple printed electronic functionalities into a single device capable of the measurement of hydrogen peroxide and total cholesterol. The single-use device employed printed electrochemical sensors for hydrogen peroxide electroreduction integrated with printed electrochromic display and battery. The system was driven by a conventional electronic circuit designed to illustrate the complete integration of silicon integrated circuits via pick and place or using organic electronic circuits. The device was capable of measuring 8 μL samples of both hydrogen peroxide (0-5 mM, 2.72 × 10 -6 A·mM -1 ) and total cholesterol in serum from 0 to 9 mM (1.34 × 10 -8 A·mM -1 , r 2 = 0.99, RSD < 10%, n = 3), and the result was output on a semiquantitative linear bar display. The device could operate for 10 min via a printed battery, and display the result for many hours or days. A mobile phone "app" was also capable of reading the test result and transmitting this to a remote health care provider. Such a technology could allow improved management of conditions such as hypercholesterolemia

A biosensor for the determination of high density lipoprotein cholesterol employing combined surfactant-derived selectivity and sensitivity enhancements

High density lipoprotein cholesterol (HDL-C) is a modifiable risk factor in cardiovascular disease and devices suitable for its determination at the point of care are critical to the future management of hypercholesterolaemia. An electrochemical biosensor for measuring HDL-C was developed. The biosensor was based on a homogeneous assay methodology for selective determination of HDL-C in combination with a printed electrochemical sensor for measuring the reduction of hydrogen peroxide at a silver paste electrode. The polyoxyethylene alkylene tribenzylphenyl ether surfactant (Emulgen B-66) was found to be capable of both the selective dissolution of HDL particles, as well as the enhanced electrocatalytic reduction of hydrogen peroxide. The resulting biosensor was shown to have a linear response to HDL-C from 0.5 to 4 mM (r2=0.998) with an average r.s.d. of 7%. The biosensor was also used to analyse HDL-C in thirteen serum samples and had good agreement with a commercial spectrophotometric precipitation-based assay (r=0.7222; p < 0.058)

The evolution of selective analyses of HDL and LDL cholesterol in clinical and point of care testing

Cardiovascular disease is a leading cause of death worldwide and is caused by the build up of atherosclerotic plaques in the vasculature. It is now well established that the formation of these plaques is closely related to levels of both high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol. Thus, the importance of the effective measurement of these is critical for the improved diagnosis and management of atherosclerosis. This review discusses the emergence of methodologies for the selective determination of both LDL and HDL cholesterol. It begins with an explanation of the first methodologies based on ultracentrifugation and precipitation techniques, the development of reference methods, through to the emergence of methodologies suitable for routine laboratory use, followed by the development of professional use, point of care technologies. Finally, the current status of selective tests for cholesterol based on biosensor methodologies is reviewed and the potential for application in consumer diagnostics is discussed. © 2013 The Royal Society of Chemistry

Measurement of total cholesterol using an enzyme sensor based on a printed hydrogen peroxide electrocatalyst

© 2016 The Royal Society of Chemistry. Cholesterol is a major modifiable risk factor in cardiovascular disease and can be effectively managed by a combination of medication and monitoring. There continues to be a need for new point-of-care diagnostics to measure lipid panels, including total cholesterol. Enzyme assays based on the generation of hydrogen peroxide have been very effective in this regard. This work demonstrates the application of printed electrochemical sensors to the measurement of total cholesterol in serum. The assay uses the surfactant Triton X-100 to provide electrocatalytic enhancement of a silver paste screen-printed electrode to hydrogen peroxide, while also achieving effective solubilisation of total cholesterol from lipoprotein. The resulting biosensors employed 0.5% (v/v) Triton X-100 in PBS with 156 U mL-1 cholesterol esterase and 60 U mL-1 cholesterol oxidase. Measurement of total cholesterol in serum in the range of 0 to 10 mM had a sensitivity of 2.24 × 10-8 A mM-1, with coefficient of determination of 0.984, detection limit of at least 2 mM and average relative standard deviation of 10.8% (n = 3)