Porous silicon based potentiometric triglyceride biosensor

doi:10.1016/S0956-5663(01)00129-4

Biosensors and Bioelectronics

Volume 16, Issues 4–5, June 2001, Pages 313-317

https://doi.org/10.1016/S0956-5663(01)00129-4 Get rights and content

Abstract

A novel method for estimating triglycerides is reported. Porous silicon, prepared from p-type (100) crystalline silicon was thermally oxidized and used to immobilise lipase, an enzyme, which hydrolyses triglycerides resulting in the formation of fatty acids. This causes a change in the pH of the solution. Enzyme solution–oxidized porous silicon–crystalline silicon structure was used to detect changes in pH during the hydrolysis of tributyrin as a shift in the capacitance–voltage (C–V) characteristics. Detailed calibration of the sensor is included.

Introduction

Porous silicon has all the advantages of silicon with regards to planar technology namely miniaturization, integration of signal processing circuitry, low cost and the added advantage of more surface area and greater adsorption property. With all these benefits porous silicon is a material of choice for chemical- and biosensors. Electrochemical dissolution of crystalline silicon in hydrofluoric acid (HF) based electrolyte solutions at constant current gives a sponge-like silicon matrix with pores, which is known as porous silicon (PS) (Fourier and Derrien, 1994). Oxidized PS has been used as an insulating material in IC technology (Zielinski et al., 1997). Due to the large specific surface area (>200 m² cm⁻³) (Herino et al., 1987) and its porous structure, PS can adsorb molecules easily from its surroundings, and has been used as a gas sensor device (Motohashi et al., 1995, O'Halloran et al., 1997). The adsorption property can be exploited to great advantage in biosensors where an enzymatic reaction forms the basis of the reaction, which is to be monitored (Thust et al., 1996). It is also used as an enzyme coupling matrix for micromachined reactors in glucose monitoring systems (Laurell, 1995, Drott, 1997). Recently an interferometric biosensor based on porous silicon for detection of biomolecular complexes has been reported (Victor et al., 1997).

A conventional metal oxide semiconductor field effect transistor was modified to an ion-sensitive field-effect transistor (ISFET), and was first reported by Bergveld (1970) for biomedical applications. Caras and Janata (1980) introduced an enzyme field-effect transistor (ENFET), a new type of biosensor, which can be miniaturized and can be integrated. The problems of these biosensors are immobilization of enzyme and insufficient adhesion of the sensor membrane. As a possible solution to these problems Knoll et al. (1994) proposed etching macroscopic conical holes into the silicon to stabilize the sensitive component, which involves a complex fabrication process.

The need for easy-to-operate and sensitive biosensors is perennial. Triglyceride detection and estimation is important as high blood triglyceride levels are an indication of further heart disease (health articles, http://www.peiapathways.com/health/bw.htm). Estimation of triglyceride content in food material is important from the point of view of increased health awareness among people and stringent regulatory laws in the food industry. The commercial sensors presently used for estimating triglycerides involve complex and lengthy procedures. There is immense scope for improving sensitivity and ease of operation for such sensors. Given the possibility of using porous silicon as an adsorption material for enzymes (immobilisation) and the need for a sensitive triglyceride sensor, this work reports a potential porous silicon based potentiometric triglyceride biosensor.

Section snippets

Materials and methods

All reagents were of electronic grade. Buffers were made from analytical grade reagents and deionised water. The silicon used had a resistivity of 5–10 Ωcm. The enzyme, which is sensitive to tributyrin, was porcine pancreatic lipase bought from Sisco Laboratories, Mumbai, India. The reported specific activity of the enzyme was 40 U/mg, where 1 U is defined as 1 μM butyric acid released in 1 min with tributyrin substrate.

Characterization of the sensor for estimating the triglycerides

Typical C–V curves for different pH solutions are shown in Fig. 3. Since the C–V curves shift along the voltage axis for different pH solutions, every pH solution will have a unique U_bias value to analyze the results where U_bias is the d.c. bias applied across the device to get 60% of maximum capacitance. The corresponding U_bias values for various pH solutions were extracted and plotted against their pH values as shown in Fig. 4. This plot shows that the device is sensitive to the pH of the

Acknowledgements

The authors thank Dr N. Sundaresan for useful discussions.

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Citation Excerpt :
Potentiometric biosensors are classified further on the basis of principle, electrolyte–insulator–semiconductor capacitor (EISCAP), micromechanical and porous silica as follow The pH of the solution changes with the butyric acid produced, which is directly proportional to the concentration of tributyrin in the solution (Reddy et al., 2001; Basu et al., 2005). Fernandez et al. (2009), developed a sensitive biosensor for detection of TG concentration.
Triglycerides (TGs) are the major transporters of dietary fats throughout the bloodstream. Besides transporting fat, TGs also act as stored fat in adipose tissue, which is utilized during insufficient carbohydrates supply. TG level is below 150 mg/dL in healthy persons. Elevated TGs level in blood over 500 mg/dL is a biomarker for cardiovascular diseases, Alzheimer disease, pancreatitis and diabetes. Numerous methods are accessible for recognition of TGs, among them, most are cumbersome, time-consuming, require sample pre-treatment, high cost instrumental set-up and experienced personnel to operate. Biosensing approach overcomes these disadvantages, as these are highly specific, fast, easy, cost effective, and highly sensitive. This review article describes the classification, operating principles, merits and demerits of TG biosensors, specifically nanomaterials based biosensors. TG biosensors work ideally within 2.5–2700 s, in pH range, 6.0–11.0, temperature 25–39.5 °C and TG concentration range, 0.001–100 mM, the detection limits being in the range, 0.1 nM to 0.56 mM, with working potential − 0.02 to 1.2 V. These biosensors measured TG level in fruit juices, beverages, sera and urine samples and reused upto 200 times over a period of 7–240 days, while stored dry at 4 °C. Future perspective for further improvement and commercialization of TG biosensors are discussed.
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Nanoparticles (NPs) of commercial lipase from Candida rugosa, of glycerol kinase (GK) from Cellulomonas species, of glycerol-3- phosphate oxidase (GPO) from Aerococcus viridans were prepared, characterized and co-immobilized onto a pencil graphite (PG) electrode. The morphological and electrochemical characterization of PG electrode was performed by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) before and after co-immobilization of enzyme nanoparticles (ENPs). An improved amperometric triglyceride (TG) biosensor was fabricated using Lipase NPs/GKNPs/GPONPs/PG electrode as the working electrode, Ag/AgCl as the standard electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 2.5 s at a pH 7.0 and temperature of 35 °C. The biosensor measured current due to electrons generated at 0.1 V against Ag/AgCl, from H₂O₂, which is produced from triolein by co-immobilized ENPs. A linear relationship was obtained over between a wide triolein concentration range (0.1 mM–45 mM) and current (mA) under optimal conditions. The Lipase NPs/GKNPs/GPONPs/PG electrode showed high sensitivity (1241 ± 20 mA cm⁻² mM⁻¹); a lower detection limit (0.1 nM) and good correlation coeficient (R² = 0.99) with a standard enzymic colorimetric method. Analytical recovery of added triolein in serum was 98.01%, within and between batch coefficients of variation (CV) were 0.05% and 0.06% respectively. The biosensor was evaluated and employed for determination of TG in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia. The biosensor lost 20% of its initial activity after its continued uses over a period of 240 days, while being stored at 4 °C.
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Porous silicon based potentiometric triglyceride biosensor

Abstract

Introduction

Section snippets

Materials and methods

Characterization of the sensor for estimating the triglycerides

Acknowledgements

Sens. Actuators B

Sens. Actuators A

IEEE Trans. Biomed. Eng.

Anal. Chem.

J. Electrochem. Soc.