Determination of triglycerides with special emphasis on biosensors: A review

Determination of triglycerides with special emphasis on biosensors: A review

International Journal of Biological Macromolecules 61 (2013) 379–389 Contents lists available at ScienceDirect International Journal of Biological M...

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International Journal of Biological Macromolecules 61 (2013) 379–389

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Review

Determination of triglycerides with special emphasis on biosensors: A review C.S. Pundir ∗ , Jagriti Narang Department of Biochemistry, M. D. University, Rohtak 124001, Haryana, India

a r t i c l e

i n f o

Article history: Received 29 May 2013 Received in revised form 27 July 2013 Accepted 29 July 2013 Available online xxx Keywords: Triglycerides Biosensors Immobilization Nanomaterials

a b s t r a c t Triglycerides (TG) are transesterification product of fatty acids and glycerol and engaged in the transportation of fats. Elevated triglyceride level is associated with coronary heart disease (CAD), atherosclerosis and hypolipoprotenemia. Convenient and reproducible assay systems based on enzymes are an attractive alternative to conventional analytical methods. Triglyceride biosensors (TGBs) are based on either measurement of oxygen consumed or electron generated from splitting of H2 O2 , an ultimate product, of immobilized enzymes. TGBs work optimally within 2–900 s, between pH 6.4–8.5 and the potential 0.5–4 V. TGBs measure TG level in serum directly and can be used over a period of 14 to 168 days. This review describes the analytic characteristics of various methods available for determination of TGs with special emphasis on TGBs. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Clinical significance of TG determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TG detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Titrimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Micro method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Autoanalyzer method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Thin layer chromatographic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. HPLC method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Capillary ion-column gas chromatography method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mass fragmentographic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. Basic principles of TG biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Classifications of TG biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. DO metric TG biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. Other principle involved in DO metric based biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Electrochemical biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7.1. Membrane based TG amperometric biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. TG biosensors based on conducting polymer matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.1. Various TG biosensors based on conducting polymer matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. TG biosensors based on nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9.1. Various TG biosensors based on nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +91 9416492413; fax: +91 126274640. E-mail address: [email protected] (C.S. Pundir). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.07.026

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2.10.

3. 4.

Potentiometric TG biosensors/TG biosensors based on ion selective electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10.1. TG biosensors based on ion selective electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Need, trial and commercialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

2. TG detection methods

Triglyceride (triacylglycerol, TAG or triacylglyceride or TG) is an ester derived from glycerol and three molecules of fatty acid. A glycerol molecule has three hydroxyl ( OH) groups. Each fatty acid has a saturated/unsaturated hydrocarbon chain and a carboxyl group ( COOH). In triglycerides, the hydroxyl groups of the glycerol join the carboxyl groups of the fatty acid to form ester bonds (Fig. 1). Triglycerides, as major components of very-low-density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy source and act as transporters of dietary fat. In the intestine, triglycerides are splitted into monoacylglycerol and free fatty acids, in a process called lipolysis, with the secretion of lipases and bile, which are subsequently moved to absorptive enterocytes, cells lining the intestine.

Various methods are available for determination of triglyceride in serum or plasma. A variety of methods including chemical [3], high performance liquid chromatography [4,5], mass spectrometery [6], microtiter plate method [7], bioluminescent method [8,9] and enzymic colorimetric and fluorimetric methods [10,11] and enzyme electrode are available [12]. Most of these methods are precise and suitable for many applications, but do not satisfy the requirements for a simple, fast, accurate and specific analysis, as these are complicated, require time-consuming sample pretreatment, expensive instrumental set-up and skilled person to operate.

1.1. Clinical significance of TG determination In the human body, high levels of triglycerides in the bloodstream have been linked to atherosclerosis and the risk of heart disease and stroke. The risk can be partly accounted for by a strong inverse relationship between triglyceride level and high density lipoprotein (HDL)-cholesterol level [1]. If triglyceride level is less than 150 mg/dl, it is said to be normal; if level lies between 150 to 199 mg/dl, it is considered at the verge of hyperlipoprotenemias, and if the level is more than 500 mg/dl, it is associated with high risk of pancreatitis and above than 1000 mg/dl, it is associated with hyperlipidemia and the level above than 5000 mg/dl is associated with eruptive xanthoma, enlarged liver and spleen [2]. The present review describes the principles, merits and demerits of various analytic methods for determination of triglycerides with special emphasis on amperometric biosensors. It also deals with the applications of nanomaterials for construction of improved TG biosensors.

2.1. Chemical methods Earlier routine determinations of triglycerides were based upon estimation of the difference between the total lipids and sum of cholesterol, cholesteryl ester and phospholipids. The following chemical methods were developed. 2.1.1. Titrimetric methods Principle: A titration is a technique where a solution of known concentration is used to determine the concentration of an unknown solution. The titrimetric procedures were followed, after extraction with organic solvents and hydrolysis by NaOH. The extracts were saponified and then back titrated to assess the amount of released fatty acids, which was directly proportional to the concentration of TG [13]. Merits: Absolute determination of content, easy to use, high degree of automation attractive cost/benefit ratio, accurate and reproducible results. Demerit: The blank showed the color and the method was non specific and had interference by various biological compounds. 2.1.2. Micro method Principle: The micromethod for direct determination of blood triglyceride was based on quantitation of glycerol moiety of this molecule. Glycerol was quantified by oxidation with periodate, using colorimetric measurement of the product. Merits: A good linearity was established from 0 to 200 mg of triglyceride. A triglyceride range of 37–134 mg% was found with 7.1% standard error [14]. Demerit: The cost of the equipment required to perform these analyses is expensive. One needs an ultracentrifuge, rotor(s), fractionator, and microtiter plate reader. In addition, a computer and automated liquid pipetting devices are desirable for faster sample throughput. Secondly, the visual resolution of discrete LDL and HDL subspecies by the EQdensity gradient method is not as apparent as with native gradient gel electrophoresis (7, 9) or as with other density gradient centrifugation methods.

Fig. 1. Schematic representation of chemical structure of triglyceride (TG).

2.1.3. Autoanalyzer method Principle: The automated procedures developed for the autoanalyzer require a manual extraction into a suitable solvent and then a preliminary purification of the extract, before it is introduced into the instrument. Thus, the autoanalyzer procedures are only partly mechanized. Additionally, the assay requires a relatively high

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temperature for the alkaline hydrolysis of glycerides. Because of the harsh chemicals and solvents used, it is necessary to use special and expensive tubing, which must be changed frequently. The assay is complex and as a result the manifolds used are in most cases rather complicated. The procedure offers several distinct advantages over the same assay as conducted manually. Merits: It increases the number of tests performed by one individual in a given time period, speeds up the result. It also minimize the variation in results from one individual to another (for accuracy, coefficient of variation is reduced hence the reproducibility increases). Demerits: The various analytical steps involved in chemical estimation of serum triglyceride require a high degree of technical competence and efficiency. The chief disadvantage of chemical method is that these lend themselves poorly to generalized use in clinical laboratory. 2.2. Chromatographic methods The various chromatographic methods were used to determine triglyceride level in serum is as follows: 2.2.1. Thin layer chromatographic method Principle: It involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. In the method the triglycerides are isolated by means of thin layer chromatography. The glycerol obtained after acid hydrolysis is converted into tri-trimethyl glycerol, obtained after analysis with a gas-chromatograph – mass spectrometer equipped with on MID (multiple ion detector). A method was also developed for determination of triglycerides in small biological samples in limited number of cells. The method was based upon thin layer chromatography and molebdatophosphoric acid staining using laser densitometry [15]. Merits: The major advantage was that nanogram of triglyceride could be measured. The method was low cost assay and highly reproducible and could be used for routine lipid analysis, due to its higher sensitivity than enzymatic method. But the method is time consuming method. Demerit: Spots are often faint, and TLC is difficult to reproduce, uneven advance of solvent, front streaking, spotting 2.2.2. HPLC method Principle: HPLC procedure is based on adsorption chromatography and refractive index for detection of triglycerides. The determination of monoglycerides by HPLC has also been developed. The detection limit was 0.1 ␮g and correlation coefficient (r) was 0.999. A simple and reliable method for quantitative detection of triglycerides in serum lipoproteins and serum free glycerol by HPLC has been developed [16]. After separation of serum constituents using a new gel permeation column, a new eluent triglyceride and free glycerol (FG) were detected by online reaction using a modified enzymatic reagent. HPLC patterns showed five peaks and absolute concentrations of triglyceride in each lipoproteins fraction were calculated from corresponding peak using a calibration. Merits: The method is highly specific, because it did not include di or monoglycerides in the measurements [17] .The method was faster and cheaper [18]. This method had high sensitivity. Demerits: Costly equipments, time consuming, expertise handling. 2.2.3. Capillary ion-column gas chromatography method Principle: An accurate method based on capillary ion-column gas chromatography for determination of triglyceride was also proposed. The resulting glycerol was directly measured by gas

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chromatography (GC). This method was linear up to 37 m mol/l and GC method upto 22 m mol/l [19]. Merits: Shorter run times, cheaper columns, higher signal to noise ratio. Demerits: Only compounds with vapor pressures exceeding about 10–10 torr can be analyzed by gas chromatography mass spectrometry (GC–MS). Many compounds with lower pressures can be analyzed, if these are chemically derivatized (for example, as trimethylsilyl ethers). Determining positional substitution on aromatic rings is often difficult. Certain isomeric compounds cannot be distinguished by mass spectrometry e.g. naphthalene versus azulene, but these can often be separated chromatographically. 2.3. Mass fragmentographic method Principle: In the method, a fixed amount of a mixture of [1,1,2,2,3,3-2H5] glycerol tripalmitate and [1,1,2,2,3,3-2H5] glycerol trioleate (500 nmol) is added to a fixed amount of serum (250 ␮1) and extracted with chloroform/methanol (2:1, v/v). The triglycerides are isolated by means of thin-layer chromatography. The glycerol obtained after acid hydrolysis is converted into the trimethylsilyl derivative and the amount of unlabeled glycerol is determined from the ratio between the recordings at m/e 218 and m/e 222, obtained after analysis with a gas chromatographmass spectrometer equipped with an MID (multiple ion detector). The two ions corresponded to the peak at M–90 and M–91 in the mass spectrum of the trimethylsilyl derivative of unlabeled and (1,1,2,3,3-2H5)-labeled glycerol [20]. 2.4. Other methods In vivo determination of triglycerides secretion using radioactive glycerol in rats fed with different dietary saturated fats led to changes in plasma triglyceride (GTG), Specific radioactivity of TG was determined in unanaesthetized fasted rats after injection of 100 aC (2–3) of labeled glycerol [21]. Microtiter plate method for enzymatic determination of triglycerides (TG) was developed. The advantage of serum lipid levels included the small sample size and saving of time and cost of reagents [7]. Quantitative analysis of triglycerides using atmospheric analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry was performed. This method was more accurate other than chromatographic methods, as the latter were dependent on the quality of chromatographic resolution of the triglycerides species [6]. 2.5. Biosensors A biosensor is a device that is used for the detection of an analyte by combining a biological component with a physico-chemical detector component. It consists of 3 parts mainly, a sensitive biological element, the transducer or the detector element and associated electronics or signal processors. Biological materials such as tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids can be used as biological element. The detector element works in a physico-chemical way like optical, piezoelectrical, electrochemical or thermal that transforms the signal resulting from the interaction of the analyte with the biological element into another signal that can be easily measured and quantified [22]. The aim of a biosensor is to produce either discrete or continuous digital electronic signals, which are proportional to a single analyte or a related group of analytes [23]. Several types of transducers have been used in the development of biosensors [24] (Fig. 2). Enzyme electrodes are an attractive alternative method, due to their simplicity, fast results and sensitivity and possibility of performing analysis without any pretreatment of sample. Amperometric

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Fig. 2. General diagram of biosensor.

sensors monitor the current generated, when electrons are exchanged either directly or indirectly between a biological system and an electrode [24]. 2.5.1. Basic principles of TG biosensors Enzymatic amperometric TG biosensors are the most common device used for TG determination. These TG biosensors are based on interactions of three enzymes, lipase, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO). Two general strategies have been used for electrochemical sensing of TG by (i) measuring oxygen consumption or the amount of hydrogen peroxide produced by the enzymatic reactions. The enzymatic reaction scheme for measuring the TG level by amperometric biosensor is presented in Fig. 3. The enzymatically produced H2 O2 can be monitored by an electrochemical sensor Therefore, TG level is determined using this biosensor by measuring the output of current changes from the electro oxidation of H2 O2 . Most triglyceride biosensors reported till dates are based on multi-enzymes, wherein a biochemical reaction depends upon kinetics of the three enzymatic reactions. These biosensors can be, sometime expensive and time-consuming. Hence TG biosensors are fabricated based on bienzymic TG sensing. Triglyceride analysis is carried out based on hydrolysis of triglyceride into fatty acids and glycerol. The glycerol can then be directly oxidized by glycerol dehydrogenase with the participation of nicotinamide adenine dinucleotide (NAD+ ) as the electron acceptor, resulting in the formation of dihydroxyacetone (NADH) and hydrogen ions. The enzymatically produced NADH can be monitored by an electrochemical sensor through its re-oxidation cycle to NAD+ . Therefore, the triglyceride concentration can be determined using this biosensor by measuring the output current changes from the enzymatic oxidation reduction of NAD (Fig. 4). 2.6. Classifications of TG biosensors 2.6.1. DO metric TG biosensors Dissolved oxygen (DO) electrode uses two types of electrode preparation for amperometric determination of TG. Principle: The method was based on the principle that indirect electrochemical monitoring of NADH via it’s reaction with

oxygen by horseradish peroxidase derive amperometric signals due to oxygen depletion, which provide for a one-point kinetic analysis. Lipase hydrolyses triglycerides to glycerol and glycerol dehydrogenase catalyses the reaction of glycerol with NAD+ to produce dihydroxyacetone and NADH. NADH may be indirectly monitored amperometrically using a membrane oxygen electrode according to the reaction HRP

NADH + H+ + 1/2 O2 −→ NAD+ + H2 O Mn2+

HRP = horseradish peroxidase Under appropriate condition, the maximum rate of change in the dissolved O2 concentration was proportional to initial triglyceride concentration. 2.6.2. Other principle involved in DO metric based biosensor The biosensor measures dissolved O2 utilized in the oxidation of TG by membrane bound lipase, glycerol kinase (GK) and glycerol3-phosphate oxidase (GPO), which is directly proportional to TG concentration. Chemical reactions involved in the principle of this method were as follows: Lipase

TG + H2 O −→ Glycerol + 3 free fatty acids GK

Glycerol + ATP−→L − ␣ − glycerol phosphate + ADP L − ␣ − glycerol − 3 − phosphate + O2 → H2 O2 + DHAP DHAP = Dihydroxyacetone phosphate Supports used for immobilization of enzymes in DO metric TG biosensor: Polyvinyl chloride membrane (PVC) [25], Gelatin [26,27]Analytical parameters of DO metric TG biosensor Optimum pH range: 7.5–8.5, Linear range: 0.1–20 mM, Detection limit: 50–350 ␮M, Response time 15 min, Storage stability: 50% loss in 25 days. Table 1 provides a comparison of analytical properties of all DO metric TG biosensors.

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Fig. 3. Scheme of electrochemical reactions involved in a TG biosensor based on lipase, GK and GPO. GK = Glycerol kinase; GPO = Glycerol-3-phosphate oxidase; Chit = Chitosan.

Fig. 4. Electrochemical reactions involved in functioning of TG biosensor based on lipase and NAD+ dependent glycerol dehydrogenase . .

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Table 1 A comparison of analytical properties of all DO metric TG biosensors. Type

Type of support for immobilization

Method of immobilization

Optimum pH

Detection limit

Linear range

Response time

Storage stability

Reference

DO DO DO

PVC Gelatin NR

NR Entrapment NR

7.5 8.5 NR

0.35 mM 0.1 mM 50 ␮M

5 to 20 mM 0.1 to 4.0 mM NR

NR 15 min NR

50% loss in 25 days 50% loss in 19 days NR

[25] [26] [27] NR-Not reported

Merits: DO metric TG biosensors are easy to use, can be operated at the bedside of patient/outside, can easily and conventionally be employed without special expertise and training. Demerits: Interference due to atmospheric O2 , which make these biosensors less sensitive. 2.7. Electrochemical biosensors Electrochemical biosensors have been used mainly for the detection of TG concentration. Principle: The underlying principle for this class of biosensors is that many chemical reactions produce or consume ions or electrons, which in turn cause some change in the electrical properties of the solution, which can be sensed out and used as measuring parameter. These TG biosensors are based on generation of electrons from H2 O2 under high potential as follow: Lipase

TG + H2 O −→ Glycerol + free fatty acids Glycerol

Glycerol + ATP −→ L − ␣ − glycerol phosphate + ADP L − ␣ − glycerol − 3 − phosphate + O2

Glycerol−3

−→

H2 O2 + DHAP

phosphate oxidase

0.4 V

H2 O2 −→2H+ + O2 + 2e− The electrons thus generated from H2 O2 , are passed from solution to electrode through immobilizing matrix. These electrochemical biosensors are as follows: 2.7.1. Membrane based TG amperometric biosensors The amperometric TG biosensors were based on different artificial membrane bound enzymes as given below: Supports used for immobilization of enzymes: collagen [28], AV membrane [29], cellulose acetate (CA) [30], polyvinylchloride (PVC) [31], polyvinyl alcohol (PVA) [32] and eggshell membrane [33]. Techniques used for immobilization of enzymes: Physical entrapment, microencapsulation, adsorption, covalent binding and covalent cross-linking. Electrodes used: Rotating Pt electrode, PtAnalytical characteristics: Optimum pH range: 7.0–8.5, Optimum potential range: 0.3–0.4 V Linear range: 10–500 mg/dl, Detection limit: 0.5–280 ␮M, Response time: 2 s–15 min, Storage stability: 27–70 days. Merits: These amperometric biosensors (which is based on the proper immobilization of enzyme on suitable matrixes) offer a portable, cheap, and rapid method for the determination of TG. Due to exotic properties, it is expected that biocompatible artificial membranes could be the promising matrixes for enzyme immobilization, which could enhance the sensitivity and selectivity of biosensors for the effective detection of various biomolecules. The development of biosensors based on immobilized enzymes solved several problems such as loss of enzyme (especially if expensive), maintenance of enzyme stability and shelf life of the biosensor, and additionally to reduce the time of the enzymatic response and offer disposable devices, which can be easily used in stationary or in flow

systems. General schematic representation of a membrane based biosensor is shown in Fig. 5. Demerits: The membranes had a problem of time consuming preparation, long response time, fragility and low storage stability and above all a major problem of desorption or leaching of enzymes during washing. Table 2 provides a comparison of analytical properties of all membrane based amperometric TG biosensors. 2.8. TG biosensors based on conducting polymer matrices Conducting polymers have become the materials of choice for recent technological advances in biosensor and have been extensively reviewed by various researchers [34–37]. The choice of polymer depends on its biocompatibility, better interaction of analytes that can produce significant amplified signal, shield electrodes from interfering material and have capacity for electropolymerisation on any surface of electrode. 2.8.1. Various TG biosensors based on conducting polymer matrices Supports used for immobilization of enzymes: polyaniline/singlewalled carbon nanotubes [38]. Techniques used for immobilization of enzymes: cross-linking, covalent binding Electrodes used: Indium–tin-oxide (ITO) coated glass plate, Screen-printed electrodes Analytical characteristics: Optimum pH range: 6.4–7.4, Optimum potential range: 0.15– 0.7 V Linear range: 0–885 mg/dl, Detection limit: 0.5–50 mg/dl, Response time: 12–20 s, Storage stability: 8–13 weeks. Merits: These conducting polymers are used as supports for biomolecules, ensuing in biosensors that have improved speed,

Fig. 5. Schematic outline of amperometric membrane based biosensor.

[33] 50% loss in 70 days

2.9. TG biosensors based on nanoparticles Nanostructured metal oxides are known to have unique ability to promote faster electron transfer kinetics between electrode and the active site of desired enzyme [39–42]. Chemically modified electrodes have received extensive attention owing to their evident advantages such as high sensitivity, selectivity and stability over a wide range of solution composition, less prone to surface fouling and low over potential at which the electron transfer process occurs, compared with inert substrate electrodes [43]. Enzymatic electrochemical biosensors based on disposable transducers, have become very attractive for environmental, clinical, and food analysis over the past 20 years [44]. Nanotechnology has recently become one of the most exciting forefront fields in biosensors fabrication [45]. Recently, special attention has been devoted to the nanoparticles as novel supports to immobilize and modify biomolecules. Thermal stability, irradiation resistance, electrochemical activity, high electron communication features and flexibility to form different nanostructures are the advantages that expedite their potential wide applications in biosensors. Nanoparticles are the smallest dimension structures that can be used for efficient transport of electrons and are thus critical to the function and their high surface-to-volume ratio and tunable electron transport properties make them suitable for biosensing applications. Extensive research efforts have been focussed in the earlier period of the development of biomolecule–nanoparticle hybrid assemblies and their application to construct biosensors. Metal oxide nanoparticles such as cerium oxide (CeO2 ), zinc oxide (ZnO), tin oxide (SnO2 ), titanium oxide (TiO2 ) and zirconium oxide (ZrO2 ) have recently been used for fabrication of enzyme-based biosensors. General schematic representation of biosensor based on nanoparticles is shown in Fig. 6.

0.56 to 2.25 mM 0.28 mM 0.4 7.0 NR – not reported.

Amperometric

Pt Egg shell membrane

Amperometric

385

sensitivity and versatility in diagnostics to determine desired analytes. Added advantages are that enzyme molecules can be entrapped during electro-polymerization and also that the polymer film uniformly covers the surface of working electrodes [35,36]. Demerits: Conducting polymers have a problem of its high cost, difficulty in fabrication and processing.

NR

[32] 50% loss in 70 days 0.4 PVA

Amperometric

Pt

7.0

0.21 mM

0.56 to 2.25 mM

2s

[31] 50% loss in 40days 0.4 PVC

Amperometric FIA Amperometric

Pt

7.5

0.11 mM

0.56 to 2.25 mM

30 s

[29] [30] 25% loss in 30 days 50% loss in 25 days 2 × 10−6/ 10−3 mol/l 0.2 to 3.5 mM NR 0.4 8.5 6.5

Covalent BSA glutaraldehyde cross linking BSA glutaraldehyde cross linking glutaraldehyde cross linking Covalent coupling

5 × 10−7 mol/L 0.2 mM

NR 40 s

[28] 82% loss in 27 days 10 to 500 mg/dl 0.3 8.0

Collagen membrane AV CA Amperometric

Rotating platinum electrode (RPE) Pt Pt

NR

10 mg/dl

15 min

References Potential (V) for max. current Optimum pH Method of immobilization Type of electrode Type of support for immobilization Type

Table 2 A comparison of analytical properties of amperometric membrane based TG biosensors.

Detection limit

Linear range

Response time

Storage stability

C.S. Pundir, J. Narang / International Journal of Biological Macromolecules 61 (2013) 379–389

2.9.1. Various TG biosensors based on nanoparticles Nanoparticles used for immobilization of enzymes: cerium oxide (CeO2 ) [46], iridium nanoparticles [44], carboxylated multi wall carbon nanotubes (cMWCNTs) [47], Zinc oxide nanoparticles [48], Gold polypyrrole nanocomposite [49]. Techniques used for immobilization of enzymes: Physical adsorption, electrostatic interactions, covalent cross-linking. Electrodes used: Indium–tin-oxide (ITO) coated glass plate, glassy carbon (GC) electrode. Analytical characteristics: Optimum pH range: 6.4–7.4, Optimum potential range: 0.15– 0.7 V Linear range: 0–885 mg/dl, Detection limit: 0.5–50 mg/dl, Response time: 12–20 s, Storage stability: 8–13 weeks. Merits: Nano-particles based TG biosensors have many advantages both in terms of stability and in promoting the catalytic reduction of redox species. Additionally, the electrode is notable for its ability to inhibit the oxidation of interfering species. Nanoparticles have attracted much interest owing to their unique properties including high mechanical strength, oxygen ion conductivity, biocompatibility and retention of biological activities [50,51]. Nanoparticles have increased electroactive surface of electrode resulting in enhanced electron transport between electrolyte medium and the electrode. Nanoparticles also provide an increased electroactive surface area for loading enzyme and enhancing electron transfer.

[49]

[48]

6.5

0.1 V

20 mg/dl

50–700 mg/dl

4s

half-life of 7 months half-life of over 7 months 6s 50–650 mg/dl 20 mg/dl 0.4 V 7.5

NR – not reported.

Au Electrochemical

Pt Electrochemical

Glassy carbon

MWCNT- Cerium oxide nanoparticles Zinc oxide Nanoparticles Gold polypyrrole

ITO Electrochemical

Electrochemical

carbon

Electrochemical

Covalent cross-linking Covalent

[47] more that 55 days 25 s 1–100 mg/dl 0.5 mg/dl 0.5 V 6.4

[46] 12 weeks 20 s 50–500 mg/dl 32.8 mg/dl 0.7 V 6.5

Electrostatic interactions Physical adsorption

[44] Disposable NR 0–10 mM NR +0.15 V NR

7.4 Covalent cross-linking ITO

Polyaniline/Single walled Carbon Nanotubes Iridium Nanoparticles Cerium oxide film Electrochemical

Physical

13 weeks 12 s 50–400 mg/dl

Linear range Detection limit Potential (V) for max. current Optimum pH Method of immobilization Type of electrode

The initial impetus for advancing sensor technology came from health care area, where it is now generally recognized that measurements of blood gases, ions and metabolites are often essential and allow a better estimation of the metabolic state of a patient. In intensive care units for example, patients frequently show rapid variations in biochemical levels that require an urgent remedial action. Also, in less severe patient handling, more successful treatment can be achieved by obtaining instant assays. At present, the list of the most commonly required instant analyses is not extensive. In practice, these assays are performed by analytical laboratories,

Type of support for immobilization

3. Need, trial and commercialization

Table 3 A comparison of various TG biosensors based on conducting polymer matrices and nanoparticles.

2.10.1. TG biosensors based on ion selective electrode Supports used for immobilization of enzymes: Porous silicon [53,54], mesoporus Si matrix [55], pH FET [52], silica gel beads [56], gate surface of FET [57], Silicon nitride layer of EISCAP, cantilever beams [58]. Electrodes used: Electrolyte-insulator capacitor (EISCAP), Ionselective field effect transistor (ISFET). Analytical characteristics: Linear range: 1–30 mM, Detection limit: 0.009–5.9 mM, Response time: 2–20 min, Storage stability: 2–24 weeks. Merits: Ion selective electrode based TG biosensors have many advantages like its linear response: over 4–6 orders of magnitude of A. As it is non-destructive: no consumption of analyte, non-contaminating, short response time: in seconds or minutes. It remains unaffected by color or turbidity. Demerits: Ion selective electrodes have a problem of its precision which is rarely better than 1%. 2. Electrodes can be fouled by proteins or other organic solutes, interference by other ions. Electrodes are fragile and have limited shelf life. Table 4 provides a comparison of various TG biosensors based on ion selective electrode/potentiometric based biosensors.

Response time

Potentiometric biosensors make use of ion-selective electrodes in order to transduce the biological reaction into an electrical signal. In the simplest terms this consists of an immobilized enzyme membrane surrounding the probe from a pH-meter, where the catalyzed reaction generates or absorbs hydrogen ions. General schematic representation of a potentiometric biosensor is shown in Fig. 7. The reaction occurring next to the thin sensing glass membrane causes a change in pH which may be read directly from the pH-meter’s display. Ion-selective field effect transistor (ISFET) is a robust platform to develop biosensors. A variety of methods have been used including covalent attachment or polymer entrapment, to associate enzymes or antibodies to the gate surface of a FET. In this regard, an ISFET device of small size and low weight might be appropriate for use in a portable monitoring system, i.e., a hand-held drug monitoring system. Both the fabrication of a nanoscale device and elimination of nonspecific molecular adsorption contribute to an improvement in the limit-of-detection (LOD) and selectivity of the biosensor. Investigators have conducted extensive studies in the electronic analysis of biomolecules by monitoring the variations in the charge density using ISFETs [52]. General schematic representation of biosensor based on ion-selective field effect transistor is shown in Fig. 8.

50 mg/dl

Storage stability

2.10. Potentiometric TG biosensors/TG biosensors based on ion selective electrode

NR

References

Demerits: Nanoparticles have a problem of its high cost, difficulty in preparation and in optimizing the amount for deposition. Table 3 provides a comparison of various TG biosensors based on nanoparticles.

[38]

C.S. Pundir, J. Narang / International Journal of Biological Macromolecules 61 (2013) 379–389

Type

386

C.S. Pundir, J. Narang / International Journal of Biological Macromolecules 61 (2013) 379–389

387

Fig. 6. Schematic outline of amperometric TG biosensors based on nanoparticles .

Fig. 7. Schematic outline of potentiometric TG biosensors.

Table 4 A comparison of various TG biosensors based on ion selective electrode/potentiometric based biosensors. Type

Type of support for immobilization

Type of electrode

Detection limit

Linear range

Response time

Storage stability

Reference

Potentiometric

pH-FET’s

pH-FET’s

9 ␮g/ml

100–400 mM

2 min

[52]

Potentiometric Potentiometric Potentiometric

Porous silicon Porous silicon mesoporous Si matrix

NR EISCAP Psi sample

5.9 mM NR NR

5.9–21 mM NR <150 mg/dl

15 min NR NR

Potentiometric Potentiometric

Silica gel beads gate surface of a FET

ISFET ISFET

NR

Upto 30 mM 5–30 mM.

<5 min

Potentiometric

silicon nitride layer of the EISCAP cantilever beams

EISCAP

500 ␮M

1–7 mM

45 min

88% of the initial value after 44 days 6 months NR working time of more than 17 hours within two months 40 days 45% loss of activity after 2 weeks NR

microcantilever

10 ␮M

24–480 ␮M

20 min.

microcantilever sensor NR – not reported.

[53] [54] [55]

[56] [57] [58]

388

C.S. Pundir, J. Narang / International Journal of Biological Macromolecules 61 (2013) 379–389

Fig. 8. Schematic outline of TG biosensors based on ion-selective field effect transistor (ISFET).

where discrete samples are analyzed, frequently using the more traditional analytical techniques. The importance of triglycerides has been a subject of some debate, but recent evidence strengthens the connection between high triglycerides and heart disease In the human body, high levels of triglycerides in the bloodstream have been linked to atherosclerosis (hardening of the arteries), and, by extension, the risk of heart disease and stroke. However, the relative negative impact of raised levels of triglycerides compared to that of LDL: HDL ratios is still unknown. The risk can be partly accounted for by a strong inverse relationship between triglyceride level and-HDLcholesterol level. The American Heart Association has set guidelines for triglyceride levels [59] (http://www.americanheart.org/ presenter.jhtml?identifier=183#Triglyceride). (Table 5) There is increasing demand for a more reliable, accurate TG monitoring system. TG biosensors face many problems in different terms. Most triglyceride biosensors reported till date is based on multi-enzymes, wherein a biochemical reaction depends upon enzyme kinetics of the other enzymatic reaction. Another important problem that needs to be rectified while fabricating TG biosensors, especially potentiometric biosensors, is the effect of interferants which hamper accurate monitoring. Hence, there is an urgent need for more and better analyzers that can serve the desired purpose of monitoring TG in clinical samples, be cost effective and also be free of any disadvantages usually occurring in the present day biosensors Electrochemical biosensors give a possible means of meeting such needs provided the capabilities of such sensors can be extended to a wider range of biomolecules and more complex matrices. Several recent initiatives suggest that some new approaches to the development of potentiometric and amperometric biosensors may be effective for this purpose [60]. The current tendency is to develop miniaturized systems with known characteristics such as versatility, relatively low cost of electrochemical

Table 5 A guidelines of American Heart Association for triglyceride levels. Level (mg/dL)

Level (mmol/L)

Interpretation

<150 150–199 200–499 >500

<1.69 1.70–2.25 2.26–5.65 >5.65

Normal range, low risk Borderline high High Very high: high risk

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