Biosensing methods for determination of triglycerides: A review

Biosensors and Bioelectronics 100 (2018) 214–227

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Biosensing methods for determination of triglycerides: A review

MARK

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C.S. Pundir , Vinay Narwal Department of Biochemistry, M.D.University, Rohtak 124001, India

A R T I C L E I N F O

A BS T RAC T

Keywords: Triglycerides (TGs) Detection methods for TG TG biosensors Nano-materials Biological materials Hyperlipidemia

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.

1. Introduction Triacylglycerols or triglyceride (TGs), known as natural fats, are made up of one glycerol molecule that is joined to three molecules of fatty acids (saturated/unsaturated or both) through ester bonds. TGs are the significant transporters of dietary fats throughout the circulation system. To a great extent,both very low density lipoproteins (VLDL) and chylomicrons are made up of TGs (Toth, 2011). Besides transporting fat, these can be used as fuel, when the body's energy demand is not fulfilled by carbohydrates (Dallongeville and Meirhaeghe, 2010). The normal level of TGs in blood is less than 150 mg/dL, while 150–199 mg/dL level is considered border line high and 200–499 mg/dL as high. The elevated TGs level over 500 mg/dL) in serum is used as a biomarker for cardiovascular disease (Sarwar et al., 2007), Alzheimer disease (Burgess et al., 2006), pancreatitis (Kota et al., 2012) and diabetes, due to abnormal lipoprotein metabolism. Accumulation of TGs may also be caused, by its high rate of synthesis as well as low rate of catabolism, in which the activity of triacylglycerol lipase and β-oxidase play an important role (Tirosh et al., 2008). The decreased activity of lipase catalyzing the hydrolysis of TG into free fatty acids and glycerol may result into accumulation of TGs (Ponec et al., 1995). The high TGs level affects the particle size of low density lipoprotein (LDL) negatively. Through a complex metabolic cooperation, TGs advance the development of small, dense LDL

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particles, which are especially atheroscelerogenic (V et al., 1992). Indeed, even within the presence of normal LDL cholesterol, patients with high TGs level normally have endothelial dysfunction, due to which their blood vessels do not expand and contract properly (Lakatos and Hárságyi, 1988). Moreover, excess of TGs brings down nitric oxide levels and increment of numerous inflammatory compounds, which play a part in vascular injuries and endothelial dysfunctions (Yoshida et al., 2004). Therefore, the determination of TG in blood is very important in the clinical diagnosis and medical management of various diseases. A number of methods are accessible for determination of TG such as titrimetric method (Klotzsch and McNamara, 1990), colorimetric method (Mamoru et al., 1997), enzymic colourimetric Fossati and Prencipe 1982; Kalia and Pundir, 2004) spectrophotometric method (Mocho´n and Leyva, 1984), chromatographic methods (Brunnekreeft and Leijnse, 1986), fluorometric method (Mendez et al., 1986), nuclear magnetic resonance method (Otvos, 1999), micro method (Kaplan and Lee, 1965), mass fragmentographic method (Björkhem et al., 1982) and enzymatic centrifugal method (Grossman et al., 1976; Hearne and Fraser, 1981). Titrimetric methods are non specific and affected by various interferents present in physiological samples (Greenfield and Clift, 1975). Colorimetric methods are unreliable, non-specific and insensitive, while enzymatic methods require expensive chemicals (enzymes) and applicable in limited concentration range. However, in

Corresponding author. E-mail address: [email protected] (C.S. Pundir).

http://dx.doi.org/10.1016/j.bios.2017.09.008 Received 3 July 2017; Received in revised form 31 August 2017; Accepted 6 September 2017 Available online 09 September 2017 0956-5663/ © 2017 Elsevier B.V. All rights reserved.

Biosensors and Bioelectronics 100 (2018) 214–227

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5 min, stirred and filtered and then the filtrate is diluted to 10 mL with deionized water. An aliquot of 1.0 mL of this suspension containing the extracted lipid is added to the electrochemical cell containing 25.0 mL of PBS(Phosphate buffer saline) pH 6.5. TG level in the sample is intrapolated from the standard curve between TG concentrations and biosensor response prepared under the optimum conditions.

simple chromatographic methods, chiral separation is not possible (Parker, 1983). The advanced chromatographic methods are specific and highly sensitive, but require time consuming sample pretreatment, costly equipment and trained personnel to operate. Further, these methods are too inconvenient for small volume samples,as a sequence of steps are required for sample pretreatment and derivatization (Hou et al., 2016). The proton nuclear magnetic resonance method is slow, require skilled person and does not provide the absolute TG concentration (Braithwaite and Smith, 1999). In the micro method, the cost of equipment is very high. The visual determination of discrete LDL and high density lipoprotein (HDL) molecules by the EQ density gradient method is not as clear, as with native gradient centrifugation methods (Redfield, 1978). The fluorescent spectroscopic methods are highly sensitive and can even detect a single molecule, while fluorescencebased in vivo detection of TG is not practically applicable in clinical samples (Shireman and Durieux, 1993). In the centrifugal auto analyzer, a large number of analytical steps are required for TG determination in sera of hyper-triglyceridemia patients, which involve a high level of specialized ability and effectiveness (Kamata, 1985). Nevertheless, biosensing approach overwhelms these drawbacks, as these are specific, fast, easy, low-cost, and highly sensitive (Sadana, 2006). Recently, Nanomaterials have been used in fabrication of biosensors to enhance their analytic performance. Metal oxides nanostructures have unique capacity to advance quick electron transfer rate between electrode and the active site of desired enzyme (Hou et al., 2016). Now a days, with the progression of nanotechnology, a vital number of novel nanomaterials have been synthesized and their novel properties are being gradually discovered and applied to construct improved/advanced biosensors (Maeda, 2014; Pumera, 2014; Narwal and Singh, 2014). This article provides a comprehensive review of the state-of-the-art research activities which focus on several important metal-oxide nanostructures and nanocomposites in addition to the application of carbon nanomaterials for TG biosensing. The most commonly-used detection methods for the TG sensing are also discussed.

4. Classification of TG biosensors The TG biosensors can be classified as electrochemical biosensors, conducting polymer based biosensors, metal oxide based biosensors, mid infra red fiber optic based biosensors, flow injection analysis based biosensors and microgel based optical biosensors as given below: 4.1. Electrochemical TG biosensors The electrochemical TG biosensors can be classified according to their measurement principles: i.e., potentiometric, amperometric, impedemetric, conductometric sensors, metal oxide based biosensor, optical biosensor, flow injection analysis biosensor, microgel based biosensor and enzyme nanoparticles based biosensor. 4.1.1. Potentiometric TG biosensor Potentiometric TG biosensors are based on lipase–catalyzed hydrolysis of the TG (tributyrin) to glycerol and free fatty acid (butyric acid). Fatty acid production induces changes in the pH of reaction buffer (analyte solution), which is measured using an open circuit potential configuration (Reddy et al., 2001). 4.1.1.1. Basic principle of potentiometric biosensor. Potentiometric biosensors are based on the principle of measure of the potential difference between the reference electrode and the working electrode at zero current flow. In these biosensors, a constant potential is generated by the reference electrode, while the working electrode conveys an erratic potential which depends on the concentration of analytes (Nakazato, 2013). The change in potential at the electrode-electrolyte interface from unbalanced activities of species i in the electrolyte phase (s) and in the electrode phase (β) is calculated by the following Nernst equation:

2. TG biosensors A biosensor is an analytical device in which a biological element (enzyme, antibodies) is used to sense analyte(s) in a system (Turner, 2015; Narang and Pundir, 2017). The basic principle of TG biosensor is to generate an electric signal that is proportional to the quantity of an analyte or a series of analytes being detected (Evtugyn 2013). The development of an ideal TG sensor for detection of TGs (biomarker of cardiovascular disease, alzheimer disease, pancreatitis and diabetes) must be a hot issue for the biosensor industry (Pundir and Narang, 2013). Various processes and techniques have been used in construction of TG biosensors. Among them, the electrochemical TG biosensors have attracted the maximum attention, because of their simplicity, supreme sensitivity, specificity, rapidity and economic for routine analysis (Pundir et al., 2016; Palchetti et al., 2009).

E = E0 +

s RT ai ln ZiF aiβ

where E0 represents the potential of standard electrode, R-gas constant, T-absolute temperature, F-faraday constant, ai- species activity i, and Zi - number of moles of electron involved (Yunus et al., 2013).

4.1.1.2. Sub-classification of potentiometric TG Biosensors. Potentiometric biosensors are classified further on the basis of principle, electrolyte–insulator–semiconductor capacitor (EISCAP), micromechanical and porous silica as follow

3. Sample preparation for TG determination

4.1.1.2.1. Electrolyte–insulator–semiconductor capacitor (EISCAP) based potentiometric TG biosensors. The biosensors based on an electrolyte–insulator-semiconductor capacitor (EISCAP) show a shift in the measured CV with changes in the pH of the electrolyte. Enzyme mediated biological reactions involved changes in the pH of the electrolyte (bioanalyte solution) and an EISCAP can be effectively used for detection of biological compounds (Setzu et al., 2011). When tributyrin, a short chain TG, is hydrolyzed by lipase, it results in the production of butyric acid as a product.

In TG biosensors, triolein and tributyrin have been used as substrates for enzymes (Lipase, Glycerol Kinase and Glycerol phosphate oxidase). Both triolein and tributyrin are hydrophobic in nature, which require to be treated with the surfactant (Triton X-100) to be converted into hydrophilic and then diluted suitably in distilled water as per required concentrations. An additional constraint is the limited solubility of serum TGs in aqueous electrolytes, impeding the analysis. To solve this problem, TG sera samples are incubated with 0.22 mM Triton X100 in phosphate buffer (pH-6.5), enabling efficient sample preparation for biosensor signaling. In case of TG analysis in foods, samples (20–80 mg) are added into 8.0 mL Arabic gum solution in a polypropylene tube. The mixture is placed in a water bath at 65 °C for

Lipase

Tributyrin + water ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→Glycerol + butyric acid The pH of the solution changes with the butyric acid produced, 215

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Fig. 1. Schematic representation of fabrication of miniaturized EISCAP TG biosensor (Veeramani et al., 2014).

Fig. 2. Schematic representation of porous silicon based TG biosensor (Reddy et al., 2003).

that of sensors of bigger dimensions. Merits: The main advantage of selecting EISCAP as the transducer of biosensor is its simple preparation compared to other types of transducers such as pH-FET and the NH4-ISFET, which involves the immobilization of the enzyme molecules by cross-linking and thus ensures good reproducibility. Further, unlike ISFETs, EISCAP possesses a planar surface, where no passivation of electronic circuits is required. A simple O-ring arrangement is sufficient to seal the sensor in the measuring cell (Vemulachedu et al., 2008). Demerits: It requires 30 min post processing time. Further it is complex, since the enzyme and counter electrode are not integrated with the sensor (Vemulachedu et al., 2008). 4.1.1.2.2. Porous silicon based TG biosensors. Porous silicon (PSi) is a promising candidate for several applications, due to the ease of fabrication and controllable pore size. Furthermore, it is compatible with conventional silicon processing technology (Fig. 2). The prepared PSi had pore sizes in the range of 500–750 nm with a depth of approximately 42 µm. The results of testing PSi for hydrogen ion sensing in different pH buffer solutions revealed that the PSi had a sensitivity value of 66 mV/pH that were considered a super Nernstian value. The sensors based on PSi were considered stable in the pH

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. The EISCAP sensor necessitated the presence of a buffer for stable measurements, which limits the sensitivity of the sensor at low concentrations of the bioanalyte upto 1 mM. The EISCAP was optimized to operate at 25 °C in 0.25 mM phosphate buffer (pH 6.0) containing 1 M KCl and 1 mg lipase in 1 mM Tris–HCl buffer (pH 7.4). The developed biosensor showed sensitivity of 55 ± 0.5 mV per unit change of pH, correlation coefficients of 0.979 for blood TGs and higher storage stability of 6 months (Preetha et al., 2011). Similarly, EISCAP device was fabricated by Veeramani et al. (2014) as shown in Fig. 1, which determines the TG concentration within the clinical range, 50– 150 mg/dL. The time taken by the readout system to calibrate the sensor and to measure the TG was < 5 min. Vemulachedu et al. (2008) fabricated cubical pits based biosensor having dimensions 1500 µm × 1500 µm × 100 µm, which could hold an electrolyte amount of 0.1 µL. The pH changes in the solution could be sensed through the EISCAP sensors by monitoring the flat band voltage shift in the capacitancevoltage (C-V) characteristics taken during the course of the reaction. The reaction rate was quite high in these miniature cells, compared to 216

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ment, enabling a direct and reliable pathway for detection of biomolecules with extreme sensitivity and selectivity. FET biosensors offer the benefits of high speed, low cost, and high yield manufacturing, without sacrificing the sensitivity typical for traditional optical methods in diagnostics (Vijayalakshmi et al., 2008). Demerits: Although various types of FET-based biosensors have been developed, these still suffer from a variety of fundamental and technological problems such as impurities of the semiconductor and instability of functional groups in the sensing layer (Nakako et al., 1986). 4.1.1.2.4. Micromechanical based potentiometric TG biosensor. The oxide anchored polysilicon cantilever beams, of length 200 µm, width 20 µm and thickness 2 µm with 1.6 µm gap between the beam and the substrate were fabricated in house by surface micromachining. A doppler vibrometer, with suitable modifications, was used to detect the resonance frequency of the cantilever beams. The whole system was integrated with a computer via a software program, written in LabVIEW so as to minimize any measurement errors. The cantilever beams were immersed in the tributyrin–lipase solution and excited with a sinewave of 10Vp − p coupled with a dc-offset voltage of 100 mV. The frequency of the signal was varied from 5 to 100 kHz. The frequency at which the amplitude of vibration was maximum, considered as the resonance frequency of the beam. The resonance frequencies of the cantilever vibrating in air and water were measured as 76.4 and 48.3 kHz respectively. This cantilever sensor worked without a buffer, which improved the lower level of sensitivity to 10 μM (Fernandez et al., 2009).

Fig. 3. Cyclic voltammogram response of tributyrin with and without enzyme (Reddy et al., 2003).

range, pH 2.0–12.0. The hysteresis values of the Psi based sensors were approximately 8.2 and 10.5 mV in the low and high pH loop, respectively (Al-Hardan et al., 2016). 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. The C–V characteristics of a typical experiment with and without enzyme for a tributyrin 29.1 mM concentration are shown in Fig. 3. It was found that after adding the enzyme, there was a shift in the C–V in the decreasing direction of the voltage axis due to formation of butyric acid (Reddy et al., 2003). Another, enzyme solution-oxidized PS–crystalline silicon structure was used to detect changes in pH (2–9) during the hydrolysis of tributyrin (100–700 μM), as a shift in the capacitance–voltage (C–V) characteristics. The LOD of this sensor for TG was 1 μM (Reddy et al., 2003). Merits: The very large internal surface of porous silicon is a great advantage, as like other porous materials, it allows the bonding of active molecules over a large surface in a small volume with a sensible increase of the efficiency of the devices (Al-Hardan et al., 2016). Further, PSi may be easily prepared either in powder or wafer, depending on the specific application. This allows fabrication of devices that can be dispersed in a given medium or reused (Reddy et al., 2003). Demerits: Psi based biosensors are highly costly, time consuming, less specific and more complicated. 4.1.1.2.3. Field effect transistor (ENFET) based TG biosensors. 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. A novel method was employed for retaining the enzyme molecules at the gate surface by immobilizing the enzyme on magnetic nickel ferrite nanoparticles and applying a permanent magnet below the gate of the FET. The method was able to estimate the TG concentrations in the range, 0.1–1.5% by immobilizing a thermostable lipase on nanoparticles (Vijayalakshmi et al., 2008).

Merits: It is a non-contact, non-destructive technique, which ensures high measurement accuracy with reduced testing time. Further, the cantilever sensor works without buffer with high reliability and reproducibility. Demerits: These methods are not rapid require large time in sample analysis. 4.1.1.2.5. MEMS cantilever designs with fractal surface geometry. Surface geometry plays an important role in case of analyte sensing and RF switching using beams and electrodes. The lower level detection ability and simple structure make these cantilevers more applicable in many applications. The rectangular and stepped cantilever beam structures along with fractal surface were simulated and analyzed for TG molecular pressure 294.3 Pa. Both the rectangular and stepped microcantilever beams with fractal surface exhibit better free end or tip deflection as compare to the planner surface based beam (Fig. 4) (Parsediya and Sharma, 2015). Merits: These sensors offer high sensitivity, low cost, simple procedure, requirement of sample in µL, non-hazardous procedure and quick response (Fernandez et al., 2009). With the ability of high throughput analysis of analytes and ultra sensitive detection, this technology holds tremendous promise for the next generation of miniaturized and highly sensitive sensors (Parsediya and Sharma, 2015). Demerits: The bending response of a single microcantilever is often influenced by various undesired effects, such as thermal drift and unspecific reactions taking place on uncoated cantilever surface resulting in additional cantilever bending (Parsediya and Sharma, 2015).

An enzyme electrode for neutral lipid determination based on hydrogen ion-sensitive field effect transistors (pH-FET's) was developed. The electrode was composed of two pH-FET's with an immobilized lipase membrane on one pH-FET, and a platinum wire. The electrode was used to determine triglycerides over wide concentration ranges with response time of 2 min. Relations between signal and the logarithm of the concentration are linear over the ranges 100–400 mM triacetin, 3–50 mM tributylin and 0.6–3 mM triolein. In the case of triolein, the detection limit is 9 μg mL−1 (Nakako et al., 1986). Merits: The FET devices have electronic properties comparable to traditional metal oxide semiconductor field-effect transistors (MOSFETs) and readily respond to changes in the chemical environ-

4.1.2. Amperometric TG biosensor Amperometry is a quite sensitive electrochemical technique in which the signal of interest is current that is linearly dependent on the target concentration by applying a constant bias potential. An amperometric biosensor comprises two or three electrodes. The former consists of a reference and a working electrode. Application of the twoelectrode system to biosensors is limited, because at high current flow, 217

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Fig. 4. a) Conventional rectangular microcantilever beam based switch designs with planner surface geometry b) with fractal surface geometry c) stepped microcantilever beam based switch designs with planner surface geometry d) with fractal stepped surface geometry.

to the electrode. The direct electron transfer efficiently generated an amperometric output signal (Wollenberger, 2005). The improved sensing performance by the direct electron transfer was realized by incorporating the enzyme with metal nanoparticles, and semiconductive nanomaterials (Koopal and Nolte, 1994). The nanocrystalline metal-oxide played a vital role in the enzyme immobilization owing to its highly specific surface area, good biocompatibility and stability (Khanna, 2008).

the potential control becomes quite difficult. Instead, the third electrode is commonly introduced as an auxiliary counter electrode having a large surface area to make most of current flows, between the counter and the working electrodes, though voltage is still applied between the working and the reference electrodes (Sadeghi, 2013). There are three modes of the TG oxidation referred to as the first, the second and the third generation TG sensors, depending on the electron transfer mechanisms. Fig. 5(A) depicts the first generation sensor, where oxygen is used as a mediator between the electrode and the enzyme. The oxygen is reduced to form hydrogen peroxide in the presence of TG by flavin adenine dinucleotide (FAD), a prosthetic group of enzyme, and FADH2 redox couple. The reduction rate of the oxygen is proportional to the TG concentration that is quantified by either measuring the augmentation of hydrogen peroxide or decrement of the oxygen concentration (Das et al., 2016). On the other hand, Fig. 5(B) shows second generation biosensor having artificial electron mediators e.g., ferro/ferricyanide, hydroquinone, ferrocene, and various redox organic dyes between the electrode and the enzyme s. These mediators made the electron transfer rate between the electrode and the enzyme faster and also gave a way of getting around for a case, when limited oxygen pressure commonly observed from the first generation TG sensor (Löffler et al., 1991; Poplawski et al., 1994). In Fig. 5(C), the third generation sensor, the enzyme was directly coupled

4.1.2.1. Basic principle of an amperometric TG biosensor. In amperometric TG biosensors, H2O2 is generated from TG by the combined reaction of lipase, glycerol kinase (GK) and glycerol-3phosphate oxidase (GPO)/dehydrogenase (GlDH) in a reaction cascade. The product H2O2 decomposes at high voltages to release electrons as described by the following equation: Lipase

Triglyceride ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→Glycerol + 3Fatty acids Glycerol Kinase

Glycerol + ATP ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→Glycerol phosphate + ADP Glycerol phosphate oxidase

Glycerol phosphate + O2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→Dihydroxyacetone + H2O2 high voltage

H2O2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→2H+ + ½O2 + 2e−

Fig. 5. Mechanisms of different generations of amperometric enzyme sensors, A: 1st generation biosensors where primarily, co-substrate/co-product is used as redox indicator, B: 2nd generation biosensors where artificial redox mediator is used to relay the electrons, and C: 3rd generation biosensors where direct electron transfer between enzyme and the electrode is established to generate the response (Das et al., 2016).

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The flow of electrons i.e., current, is directly proportional to the concentration of hydrogen peroxide produced by the enzyme reactions, which in turn, is directly proportional to the concentration of the analyte (TG) (Khanna, 2008). 4.1.2.1.1. (DO) metric TG biosensors. The DO metric biosensor measured dissolved O2 utilized in the oxidation of TG by membrane bound with enzymes lipase, GK and GPO, which is directly proportional to TG concentration. An amperometric TG biosensor with a thin polyurethane bienzyme membrane containing GK and GPO, sandwiched between two dialysis membranes and mounted onto a Pt electrode in a flow-through cell was combined with pumps, autosampler and potentiostat. Complete hydrolysis of TG samples was attained within 15 min with the aid of a previously freeze-dried mix containing lipase, esterase, detergent, ATP and buffer. Twenty five samples per hour were analyzed with a linear sensor signal from 5 to 1000 μmol/L and a standard deviation of 2% (repetitive measurements) (Feldbrügge et al., 1994).

tested, only cholesterol and ascorbic acid caused 15–20% and 16% inhibition, respectively. The CA membrane based enzyme electrode was used 100–200 times over 25–70 days without any significant loss of activity, when stored at 4 °C (Minakshi and Pundir, 2008; Pundir et al., 2010; Narang et al. 2009, 2010; Yücel et al., 2014). An amperometric three-electrode system was developed for the assay of triglycerides. Two enzymes, GlDH and diaphorase were immobilized onto a collagen membrane. Serum samples were incubated with a microbial lipase in phosphate buffer containing 2,6dichlorophenol-indophenol (DCPIP). The resulting glycerol was oxidized by NAD+ in the presence of GlDH; the NADH produced was then oxidized by DCPIP. The anodic current generated by oxidation of the reduced form of the DCPIP at the surface of the working electrode at 0.300 V was measured. Serum TGs ranging from 10 to 500 ng/dL were easily assayed in < 15 min using only 25 pL of sample (Schoemaker et al., 1997). Merits: TG biosensors with semipermeable membranes enhance the dynamic range along with sensitivity and selectivity. The semipermeable membrane encapsulates the enzyme as well as provides a microenvironment conducive to maintaining the enzyme's viability, thereby improving the stability of TG biosensors. Demerits: It is difficult to maintain the membrane and there are more chances of contamination due to its regular removal and attachment onto electrode.

An another method is described for preparation of a dissolved oxygen meter (make Aqualytic, Germany) based triglyceride biosensor employing a polyvinyl chloride (PVC) membrane bound lipase, GK and GPO. The biosensor showed optimum response within 10–15 s at pH 7.5 and 39.5 °C. A linear relationship was obtained between the TG concentration from 5 mM to 20 mM and oxygen consumed (mg/L). The biosensor was employed for determination of TG in serum. The within and between batch coefficient of variation (CV) were < 2.18% and < 1.7% respectively. The limit of detection (LOD)of the biosensor was 0.35 mM. A study of interference revealed that ascorbic acid, cholesterol and bilirubin caused 13%, 15%, and 12% interference, respectively (Bhambi et al., 2006). A TG measurement system was developed based on the indirect electrochemical monitoring of NADH via its reaction with oxygen by horseradish peroxidase. The biosensor showed optimum response at pH 7.5 and 33 °C within 300 s. A linear relationship was obtained between TG concentration from 0.05 to 0.3 mM and oxygen consumed (mg/L). LOD of biosensor was 0.05 mM (Kelly et al., 1984). Merits: These biosensors are easy to use and can be operated at the bedside of patient/outside, the laboratory and employed without special expertise and training. Demerits: Interference due to atmospheric O2, which make these biosensors less sensitive. 4.1.2.1.2. Membrane based amperometric TG biosensors. Four types of membrane based amperometric TG biosensors were constructed by mounting cellulose acetate (CA), polyvinyl acetate (PVA), PVC membranes and egg shell and gelatin membrane bound lipase, GK and GPO onto a Pt or Au (in case of egg shell membrane only) or glass carbon (GC) (in case gelatin membrane) electrode to construct working electrode and then connecting it to Ag/AgCl as reference electrode and Pt wire as auxillary electrode through potentiostat. The biosensors measured the electrons generated from H2O2 under a potential of 0.4 V, which in turn was generated from triolein/TGs by co-immobilized lipase, GK and GPO. The concentration of triolein/TG was directly proportional to the current measured. The enzyme electrode showed optimum response when operated between 25 and 35 °C in 0.1 M sodium phosphate buffer, pH 6.5–7.5 for 30– 40 s. A linear relationship was obtained between triolein concentration ranging from 0.2 to 3.5 mM and amount of current (mA). LOD of biosensor was 0.11–0.28 mM. The level of TG in serum of healthy and persons suffering from cardiovascular disease and pancreatitis was in the range 53.2–174.5 and 189–990 mg/dL, respectively. The analytical recovery of added triolein was between 85.2–94.5%. Within batch and between batches coefficient of variation (CVs) were < 2.14–8.0% and < 3.48–5.85%, respectively. A good correlation (r = 0.91–0.99) was found between serum TG values obtained by commercial enzymic colorimetric and present method. Among the various serum substances

4.1.3. Impedimetric/conductometric technique based TG biosensor Polyaniline nanotubes (PANI-NT) based film electrophoretically deposited onto indium–tin–oxide (ITO) coated glass plate was utilized for covalent immobilization of lipase, via glutaraldehyde, for TG detection using impedimetric technique. The fatty acid molecules produced during TG hydrolysis resulted in the change of charge transfer resistance (RCT) of PANI-NT film with varying TG concentration. The Lipase/PANI-NT/ITO bioelectrode showed a linearity as 25– 300 mg/dL, sensitivity as 2.59 × 10−3 K Ω−1 mg/dL, response time as 20 s and regression coefficient as 0.99. A low value of apparent Michaelis–Menten constant (Km) ∼ 0.62 mM indicated high enzyme affinity to tributyrin. The bioelectrode detected TG in sera (Dhand et al., 2009). Merits: It is the most powerful method, due to the ability of EISbased sensors to get integrated more easily into multi-array or microprocessor-controlled diagnostic tools (Rushworth et al., 2012). Demerits: The limitations of the EIS technique are the several requirements to obtain a valid impedance spectrum. Theoretically, there are three basic requirements for AC impedance measurements: linearity, stability, and causality. The accuracy of EIS measurement depends not only on the technical precision of the instrumentation but on the operating procedures also. 4.2. Conducting polymer based TG biosensor Conducting polymer (CP)-based electrochemical biosensors have gained great attention, such as biosensor platforms are easy and costeffective to fabricate, and provide a direct electrical readout for the presence of biological analytes with high sensitivity and selectivity. CP materials themselves are both sensing elements and transducers of the biological recognition event at the same time, simplifying sensor designs (Aydemir et al., 2016). A nanocomposite film comprising of polyaniline (PANI) and single walled carbon nanotubes (SWCNT) was fabricated onto ITO coated glass plate using electrophoretic technique. The co-immobilization of GlDH and lipase was done via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry to explore its application for TG (tributyrin) sensing. The response studies was carried out using linear sweep voltammetry (Speiser, 2007) 219

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an ITO coated glass substrate. Lipase and GlDH were immobilized onto this electrode to construct an amperometric biosensor for the lipid(tributyrin). The lipase hydrolyzed tributyrin to form glycerol, which was oxidized by GlDH to form NADH. The current measured (best at a working voltage of 0.35 V vs. Ag/AgCl) was proportional to tributyrin concentration in the range, 0.25–4 mg mL−1. The LOD of biosensor was 155 µg mL−1, and response time was 45 s. The sensor was fairly stable, as its activity was reduced to 85% within a period of 11 weeks. This bioelectrode measured TGs in sera (Solanki et al., 2016).

revealing that Lipase-GDH/PANI-SWCNT-TB/ITO bioelectrode could detect tributyrin in the range of 50–400 mg/dL−1 with low apparent Km constant of 1.138 mM, improved response time of 12 s, high sensitivity as 4.28 × 10−4 mA mg−1 dL and storage stability of about 13 weeks (Dhand et al., 2010). An electrochemical TG biosensor was developed by depositing lipase, GK and GPO) onto polymer matrices through electrostatic force and stabilized further via crosslinking. The biosensor had a detection range of 15–200 mg/dL of micellar TG. It measured TG level in sera of patients suffering from skin ailments (Jeong et al., 2014). Merits: The biosensor has qualities such as ease of preparation, stable adhesion,long electrode lifetime, as well as suitable spatial, electrostatic and chemical microenvironments constructed on the electrode surface. Demerits: Homogeneous dispersion of conducting polymer is a critical challenge. These are chemically instable and also suffer from poor processibility.

4.3.5. Nickel oxide–chitosan/zinc oxide/zinc hexacyanoferrate film based TG biosensor An amperometric TG biosensor was fabricated based on coimmobilization of lipase, GK and GPO onto nickel oxide nanoparticles (NiONPs)–CHIT nanocomposite adsorbed onto zinc oxide/zinc hexacyanoferrate (ZnO–ZnHCF) hybrid film electrodeposited on the surface of an Au electrode. The biosensor showed optimum response within 4 s at pH 6.0 and 35 °C, when polarized at + 0.4 V against Ag/ AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dL with sensitivity of 0.05 μA/mg/dL. The biosensor determined TG in serum. LOD of the biosensor was 10 mg/dL. The biosensor was evaluated with 95–96% recovery of added triolein in sera and 2% and 3% within and between batch CVs, respectively. There was a good correlation (r = 0.99) between serum TG values by standard enzymic colorimetric method and the present method. The biosensor lost 50% of its initial activity after its 100 uses over a period of 180 days, when stored at 4 °C (Narang et al., 2013).

4.3. Metal oxides based TG biosensor Recently the nanostructured metal oxides (NMOs) have become important materials for biosensors, as these provide an effective surface for biomolecule immobilization with desired orientation, better conformation and high biological activity resulting in enhanced sensing characteristics. These interesting NMOs are expected to find applications in a new generation of miniaturized, smart biosensing devices (Solanki et al., 2011). 4.3.1. Sol-gel derived SiO2-CeO2 nanocomposite based TG biosensor Sol-gel derived silica (SiO2) film fabricated onto ITO coated glass substrate was used for self-assembly of cerium oxide (CeO2) nanoparticles to fabricate Nano CeO2-SiO2 nanocomposite. The NanoCeO2SiO2/ITO electrode was utilized to immobilize lipase via physical adsorption for TG detection. The biosensor showed the linearity from 50 to 400 mg dL−1 with high sensitivity (2.28 mA mg−1 dL) and stability. A low apparent Km (~ 2.4 mM) indicated high enzyme affinity to tributyrin (Gupta et al., 2014).

4.3.6. CHIT and ZnO nanoparticles composite film based TG biosensor An amperometric TG biosensor was developed based on covalent co-immobilization of lipase, GK and GPO onto CHIT and ZnO nanoparticles (ZnONPs) composite film electrodeposited onto Pt electrode. The sensor showed optimum response within 6 s at 0.4 V, pH 7.5 and 35 °C. A linear relationship was obtained between a wide triolein concentration range (50–650 mg/dL) and current (mA) under optimum conditions. The biosensor showed high sensitivity, LOD (20 mg/dL) and good storage stability (half-life of 7 months at 4 °C) (Narang and Pundir, 2011).

4.3.2. Nanostructured cerium oxide based TG biosensor A TG biosensor was constructed by immobilizing lipase onto sol– gel derived nanostructured cerium oxide (Nano-CeO2, diameter: 35 nm) film deposited onto ITO coated glass plate, which exhibited linearity, detection limit and shelf life as 50–500 mg/dL, 32.8 mg/dL and 12 weeks, respectively. The apparent Km was 22.27 mg/dL (0.736 mM) for lipase/nano-CeO2/ITO bioelectrode indicating high affinity of lipase with tributyrin. The biosensor measured TG concentration in sera (Solanki et al., 2009). 4.3.3. Electrochemically reduced graphene oxide (ERGO) based TG biosensor Recently, reduced graphene oxide (RGO) has triggered research on electrochemical biosensing due to its high electron mobility, excellent electrochemical properties and high surface area. Graphene oxide (GO) was synthesized via modified Hummer's method (Batra et al., 2016; Narwal and Pundir 2017b). A uniform thin film of RGO was electrodeposited onto ITO electrode using chronoamperometric technique. GlDH and lipase were co-immobilized onto ERGO/ITO electrode via carbodiimide chemistry to prepare bio electrode (Lip-GlDH/ERGO/ ITO) for sensing TG. The fabricated electrode could detect tributyrin in the range, 50–300 mg/dL with high sensitivity and a low apparent Km ~ 0.152 mM indicating high enzyme affinity of bioelectrodes towards tributyrin (Bhardwaj et al., 2015).

4.3.7. Inkjet-printed Au/PEDOT-PSS nanocomposite based TG biosensor A highly sensitive disposable amperometric TG biosensor was constructed based on co-immobilization of lipase, GK and GPO onto Au/PEDOT-PSS nanocomposite inkjet-printed on screen-printed carbon electrodes (SPCEs) and one-step chemically synthesized Au/ PEDOT-PSS nanocomposite deposited by inkjet printing to produce in nanorod structures on SPCE surface. The optimal preparation conditions of AuNPs were 0.015 M EDOT and 6.25 mM gold precursor. SPCE electrode with 5 layers of inkjet printed, Au/PEDOT-PSS nanocomposite provided optimum performances, when operated with the operating potential of 0.4 V in 0.1 M sodium phosphate buffer pH of 7.0. The biosensor showed a wide dynamic range (0–531 mg/dL), moderate sensitivity (2.66 μA/mM), modest response time (30 s), low LOD (7.88 mg/dL), low interference, good reproducibility and satisfactory life time (40% loss of response current after 30 days of storage at 4 °C). Thus, the inkjet-printed Au/PEDOT-PSS nanocomposite on SPCEs was a promising candidate for TGs determination (Phongphut et al., 2013). Merits: The electrochemical responses were significantly enhanced by gold nanoparticles (AuNPs).

4.3.4. CHIT-nano-ZrO2/ITO based TG biosensor A nanocomposite was prepared from CHIT and zirconium oxide nanoparticles (nano-ZrO2) and then electrophoretically deposited on

4.3.8. Magnetic nanoparticles/zinc oxide/zinc hexacyanoferrate film based TG biosensor An effectual electrochemical TG biosensor was developed by 220

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posite based films electrochemically deposited onto ITO coated glass plate were utilized for covalent immobilization of lipase, via EDC and NHS chemistry for TG detection using amperometric technique. The electrochemical response of lipase/PtNP/PPY/ITO multilayered bioelectrode exhibited linearity, LOD and shelf life as 50–500 mg/dL, 25 mg/dL and 9 weeks, respectively. The apparent Km was 18.54 mg/ dL (0.89 mM) indicating high affinity of lipase with tributyrin. The biosensor was employed to detect TG concentration in sera samples (Chauhan et al., 2013). Merits: Metal oxides provide an effective surface for biomolecule immobilization with desired orientation, better conformation and high biological activity resulting in enhanced sensing characteristics. Nanostructured metal oxides with unique optical, electrical and molecular properties along with desired functionalities and surface charge properties provide interesting platforms for interfacing biorecognition elements with transducers for signal amplification. Demerits: It is difficult to control the synthesis of nanoparticles to form modulatory structure design.

covalently co-immobilizing enzymes onto modified glassy carbon electrode (MNP-CHIT/ZnO-ZnHCF/GCE). The LOD and working range of the biosensor were 10 mg/dL and 10–1000 mg/dL respectively. The modified electrode was anti-interferants for electrochemical sensing of TG (Narang et al., 2014). Merits: This fabrication of TG biosensor was of significant interest, because of its signal amplification in the form of current and excellent storage stability 4.3.9. Nanoporous gold biocomposite based TG biosensor A glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). The linearity for tributyrin and olive oil concentrations ranged from 50 to 250 mg/dL and 10 to 200 mg/dL respectively. Apparent Km for tributyrin was 10.67 mg/dL and LOD was 2.68 mg/dL. Enzyme electrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life (Wu et al., 2014). Merits: Due to highly conductive, porous, and biocompatible threedimensional structure, NPG was suitable for enzyme immobilization. These properties along with a long self-life made the lipase/NPG/GCE bioelectrode an excellent choice for the construction of TGs biosensor.

4.4. Mid-infrared fiber optic based TG biosensor Krug and Kellner (1993) tried mid-infrared fiber optic based TG biosensor for determination of TGs after the extraction of the sample's lipid content into an organic solvent. An infrared (IR) spectrum of the organic phase was recorded using a 10 cm piece of an uncoated chalcogenide fiber, which was incorporated into a flow cell. The characteristic absorption bands of the lipid constituent TGs were evaluated. The method covered the biological and clinical interesting range and the LOD for the lipid constituents varied from 1 to 4 mmol/ L. Merits: These are not subjected to electrical interferences. The absence of electrical connectors makes them safer than electrochemical biosensors. Demerits: No reference electrode is used, but a reference source is useful.

4.3.10. Prussian blue modified TG biosensor A Prussian blue (PB) modified screen-printed electrode (SPE) was selected as support for the two immobilised-enzyme systems due to their higher operative stability. The lipase activity against triacylglycerols was measured using an amperometric biosensor based on GlDH/ NADH oxidase. The effect of coenzymes (flavin mononucleotide, FMN at 1 mM and NAD+ at 2 mM), pHs (phosphate buffer pH 6–8, Tris buffer pH 8–10) response time and storage stability were evaluated and optimized (Rejeb et al., 2007). 4.3.11. Iridium nano-particle catalyst based TG biosensor An iridium nano-particle modified carbon based biosensor was prepared to detect TG. Glyceryl tributyrate was chosen as a substrate for evaluation of this TG biosensor in bovine serum and human serum. A linear response to glyceryl tributyrate in the concentration range, 0– 10 mM and a sensitivity of 7.5 nA mM −1 in bovine serum and 7.0 nA mM−1 in human serum were observed. The incorporation of a selected surfactant and an increase in the incubation temperature enhanced the performance of this biosensor. The experimental results demonstrated that this iridium nano-particle modified working electrode based biosensor provided a relatively simple means for the accurate determination of TG in serum (Liao et al., 2008).

4.5. Flow-injection analysis based TG biosensor A flow-injection enzymatic electroanalytical system for determination of TG was based on enzymatic reactions in capillary followed by electrochemical detection. The fitted model, per RSM, showed that optimum conditions of the system were 2 mM ATP in 0.1 M carbonate buffer (pH 11.0), flow rate of 0.18 mL/min, temperature of 35 °C, 20 µL of sample injection, and applied voltage of 0.650 V. The proposed biosensing system consisting lipase, GK, and GPO exhibited a flow-injection analysis peak response of 2.5 min and a LOD of 5 × 10−5 M glycerol (S/N = 3) with acceptable reproducibility (CV < 4.30%). It showed linearity ranging from 10−3 to 10−2 M for TG. The capillary enzyme reactor was stable up to 2 months in continuous operation, and analyzed 15 samples per hour (Wu and Cheng, 2005). Merits: FIA biosensors are simple, flexible and economic. These involve automation in sample preparation and detection, rapid start-up and shutdown times. Demerits: Require large amount of samples and chemicals.

4.3.12. AuPPy nanocomposite and poly (indole-5-carboxylic acid) based TG biosensor An amperometric TG biosensor was developed based on covalent co-immobilization of lipase, GK and GPO onto Au polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at + 0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dL. LOD of biosensor was 20 mg/dL. The biosensor was evaluated with 91–95% recovery of added triolein in sera and 4.14% and 5.85% within and between batch CVs, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. The biosensor lost 50% of its initial activity after its 100 uses over 7 months, when stored at 4 °C (Narang et al., 2012).

4.6. Microgel based optical device for TG biosensor The lipase-modified pH-responsive poly N-isopropylacrylamide-based microgels were synthesized for construction of an optical device for TG determination and loaded with lipase. The chemical synthesis of microgel and further fabrication of optical device by sandwiching the lipase microgels between two thin Au layers is shown in Fig. 6. The device's response depended on the TG concentration, demonstrating its potential application as a TG biosensor. The increase in triolein (act as a model for TG) concentration from 1 mg mL−1 to 7 mg mL−1 led to l3 blue-shifts from 39 nm to 175 nm and saturated at 7 mg mL−1 (Zhang et al., 2015).

4.3.13. Platinum nano particles (PtNPs) and polypyrrole(PPY) nano composite based TG biosensor The PPY/PtNP/PPY/ITO (PtNP/PPY/ITO) multilayered nanocom221

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Fig. 6. Schematic representation of functioning of microgel based optical biosensor for triglyceride determination.

Fig. 7. Schematic representation of fabrication of Lipase NPs/GKNPs/GPONPs/PG electrode and electro-chemical reactions involved in its response measurement.

good correlation (R2 = 0.99) was obtained between TG values in sera as measured by the present bionanosensor and the enzymic colorimetric method The ENPselectrode was used 180- times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C (Pundir and Aggarwal, 2017). An improved amperometric TG biosensor was designed in our laboratory by co-immobilizing mixture of lipase NPs, GKNPs and GPONPs onto pencil graphite electrode (PGE) (Fig. 7). This ENPs/ PGE based biosensor showed optimum response at 0.1 V against Ag/ AgCl, within 2.5 s at a pH 7.0 and 35 ⁰C. A linear relationship was observed between a wide triolein concentration range (0.1–45 mM) and current (mA). The ENPs/PG electrode showed high sensitivity (1241 ± 20 mA cm−2 mM−1), a lower LOD (0.1 nM) and good correlation coefficient (R2 = 0.99) with a standard enzymic colorimetric method. Analytical recovery of added triolein in serum was 98.01% & within and between batches CV was 0.05% and 0.06% respectively. The biosensor measured TG in the serum of apparently healthy subject and persons suffering from hypertriglyceridemia. The ENPs electrode lost only 20% of its initial activity after its continued uses over a period of 240 days, while being stored at 4 °C (Narwal and Pundir, 2017a).

Merits: N-isopropylacrylamide-based microgels are easily synthesized photonic material and stimuli responsive polymer. Demerits: Microgels are thermally instable. 4.7. Enzyme nanoparticles based amperometric TG biosensors In TG biosensors, direct immobilization of enzymes onto nanocomposites might bring about their denaturation, prompting to the loss of their activity and stability. This problem was overcome using ENPs instead of native enzyme molecules. ENPs are the aggregates of enzyme molecules in the nanoscale, which show their exceptional physicchemical properties (Pundir, 2015; Narwal et al., 2017). An amperometric triglyceride bionanosensor was fabricated by immobilizing covalently the nanoparticles of commercial lipase, GK and GPO onto Au electrode for improved amperometric determination of TG in blood. The bionanosensor exhibited optimum current at 1.2 V within 5 s, at pH 6.5, 35 °C with LOD as 1.0 µg/mL and working range from 10 to 500 mg/dL. Analytical recovery of added triolein in sera (50 and 100 mg/dL) was 95.2 ± 0.5% and 96.0 ± 0.17% respectively. Within and between batch CV were 2.33% and 2.15% respectively. A 222

Psi based electrode

Mesoporous PSi matrix

EISCAP Silicon nitride layer of the EISCAP Microcantilever sensor

EISCAP Miniaturized EISCAP

Crystalline silicon based miniaturized EISCAP Psi based electrode

Gate surface of a FET (ISFET). < 5 min LIP/Pt wire (pH-FET) LIP/GK/GPO/PUM

LIP/GK/GPO/CA

LIP/GK/GPO/HRP/PVA LIP/GK/GPO/PVC LIP/GDH/Oxygen electrode LIP/GK/GPO/Egg shell membrane/Au LIP/GK/GPO/gelatin membrane/GCE LIP/GK/GPO/POD/ glass beads

Potentiometric

Potentiometric

Potentiometric Potentiometric

Potentiometric Potentiometric

Potentiometric

Potentiometric

DO

DO DO DO

223

GDH/Diaphorase/ Collagen membrane LIP/GK/GPO/HRP/PVC

LIP/GK/GPO LIP/PANI-NT/ITO

LIP/GDH/PANI/ SWCNT/ITO LIP/NanoCeO2-SiO2/ ITO Lip/nano-CeO2/ITO LIP/GDH/TB/ERGO/ ITO LIP/GK/GPO/CHITnano-ZrO2/ITO NiONPs-CHIT/ZnO-

Amperometric

Amperometric Impedimetric

Amperometric

Amperometric

Amperometric

Amperometric Amperometric

Amperometric

Amperometric

LIP/GK/GPO/HRP/PVC

Amperometric

Amperometric

Amperometric

Amperometric

Potentiometric DO

Potentiometric

Potentiometric

Type of transducer

Type of biosensor

NR 0.3

Covalent Binding Covalent Binding

Covalent Binding

0.4

0.35

NR

Covalent Binding

Covalent Binding

NR

0.3 NR

0.4

0.3

NR

35

NR

NR NR

NR

NR

NR NR

25

NR

35

40

40

− 0.7 NR

35

25 39.5 33

25

NR NA

0.4

0.4 0.4 NA

0.4

NR NA

NR

NR

− 0.01 NR

NR

NR NR

NR

NR NR

0.2

NR − 0.02

0.01

NR NR

NR

NR

− 0.01 NR

Optimum Temp (°C)

Working Potential (V)

Covalent Binding

Covalent Binding Covalent Binding

Crosslinking

Adsorption

Crosslinking

Crosslinking

Crosslinking

Adsorption

Crosslinking Crosslinking NA

Crosslinking

Physical adsorption Physical adsorption Adsorption Adsorption

Covalent Binding

NR Covalent Binding

NR

Physical adsorption Physical adsorption NR Covalent Binding

Method of immobilization

Table 1 Comparison of analytic characteristics of various triglycerides biosensors.

6.0

7.4

NR 7.8

7.4

NR

NR NR

7.5

8

7.0

7.5

7

7.0

7 7.5 7.5

6.5

NR 7.8

NR

7

7

6.0 7.4

NR

7.0 7.0

7.0

7.0

Optimum pH

0.0001– 0.05 0.56–7.91

0.56–5.65 0.56–3.39

0.565–4.52

0.56–4.52

0.17–2.26 0.28–3.39

0.56–2.25

0.11–5.6

0.1–15

0.20–1.38

0.14–1.125

0.56–2.25

0.56–2.25 5–20 0.05–0.3

0.2–3.5

0.6–3 0.005–1

5–30

5–15

5

0.1–18 0.56–1.68

0.024–0.48

5–15 1–7

< 0.56

5.9–21

Linearity (mM)

0.11

0.000155

0.37 0.56

0.565

0.56

0.17 0.28

0.11

0.1

0.1

0.20

0.14

0.28

0.21 0.35 0.05

0.2

0.00009 0.005

5

5

NR

0.1 0.56

0.01

5 5

NR

NR

Detection limit (mM)

4

45

NR NR

12

12

NR 20

30

900

NR

1200

NR

10

2 10 300

40

120 90

< 900

900

30

1800 300

1200

NR 2700

NR

900

Response time (s)

NR NR

NR 0.05 μA/mg/dL

NR NR

NR

2.28 mA mg−1 dL NR NR

NR

NR NR

NR

2.8

2.2

NR

2.21

2.14

4.13 NA NA

4

NR NA

NR

NR

NR

NR NR

NR

NR NR

NR

NR

RSD (%)

NR 2.59 × 10−3 K Ω−1 mg/dL NR

NR

NR

NR

NR

NR

NR

NR NR NR

NA

NR NA

57.6 mV/s

30 mV/pH

33 mV/pH

55 mV/pH 35.25 mV/pH

NR

30 mV/pH 58 mV/pH

NR

30 mV/pH

Sensitivity

50% in 180

Solanki et al. (2009) Bhardwaj et al. (2015) Solanki et al. (2016)

Gupta et al. (2014)

Dhand et al. (2010)

Jeong et al. (2014) Dhand et al. (2009)

Chauhan et al. (2013) Schoemaker et al. (1997) Narang et al. (2009)

Minakshi and Pundir (2008)

Yücel et al. (2014)

Vijayalakshmi et al. (2008) Nakako et al. (1986) Feldbrügge et al. (1994) Minakshi and Pundir (2008) Pundir et al. (2010) Bhambi et al. (2006) Kelly and Christian (1984) Narang et al. (2010)

Basu et al. (2005) Fernandez et al. (2009) Fernandez et al. (2009) Preetha et al. (2011) Veeramani et al. (2014) Vemulachedu et al. (2008) Reddy et al. (2003)

Setzu et al. (2011)

Reddy et al. (2001)

Ref.

Narang et al. (2013) (continued on next page)

85% in 77 days

84 60 days

NR

91

No loss in 40days NR NR

200 times over 70 days 38.9% in 30 days 50% activity after 150 regular uses 50% loss after 200uses 18% in 27days

150 times over 25 days 50% in 50 days NA NA

45% after 2 weeks NR NA

NR

NR

NR NR

NR

< 17 h within two months NR NR

180 days

Storage stability at 4 °C

C.S. Pundir, V. Narwal

Biosensors and Bioelectronics 100 (2018) 214–227

LIPNPs/GKNPs/ GPONPs/Au LIPNPs/GKNPs/ GPONPs/PGE

Amperometric

224

Physical Adsorption

Covalent Binding

Covalent Binding

Covalent Binding

NR

Covalent Binding

Covalent Binding

Covalent Binding

Covalent Binding

0.1

1.2

0.65

NR

NA

0.4

0.1

NR

NR

NR

0.4

Covalent Binding

Covalent Binding

0.4

Working Potential (V)

Covalent Binding

Method of immobilization

35

35

35

NR

37

35

35

NR

NR

NR

NR

35

Optimum Temp (°C)

7

6.5

11

8.5

9

7.5

6.5

NR

NR

NR

NR

7.5

Optimum pH

0.1–45

0.11–5.65

10–100

0.002–1

0.04–2.0

0.56–5.65

0.56–7.91

0–10

0.56–2.83

0.11–11.3

0–6

0.56–7.35

Linearity (mM)

5 2.5

10−7 mM

NR

NR

NA

NR

4

NR

NR

NR

30

6

Response time (s)

0.001

0.05

0.0005

14

0.28

0.23

0.1

0.03

0.11

0.09

0.23

Detection limit (mM)

NR

NR

NR

NR

NR

NR

NR

NR

NR

2

2.8

NR

4.14

NR

7.5 nA mM−1 NR

NR

NR

NR

NR

2.66 μA/mM

NR

NR

RSD (%)

NR

Sensitivity

20% in 240 days

50% in 90 days

NR

8.4% in 1500 samples 30 days

50% loss after 100 uses over 210 days 270 days

NR

NR

NR

50% in 210days 40% loss in 30days

days

Storage stability at 4 °C

Chauhan et al. (2013) Schoemaker et al. (1997) Compagnone et al. (1998) Wu and Cheng (2005) Pundir and Aggarwal (2017) Narwal and Pundir (2017a, 2017b)

Narang et al. (2012)

Liao et al. (2008)

Wu et al. (2014)

Narang et al. (2014)

Narang and Pundir (2011) Phongphut et al. (2013)

Ref.

DO – Deoxymetric; LIP – Lipase; GK – Glycerol Kinase; GPO – Glycerol-3-Phosphate Oxidase; PVC – Polyvinyl Chloride; GDH – Glycerol Dehydrogenase; PUM – Poly Urethane Membrane; FIA – Flow Injection Analysis; PVA – Polyvinyl Alcohol; Psi – Porous Silicon; EISCAP – Electrolyte Insulator Semiconductor Capacitors; FET – Field Effect Transistors; ISFET – Ion Selective Field Effect Transistors; CA – Cellulose Acetate; GCE – Glassy Carbon Electrode; HRP – Horse Radish Peroxidase; PANI-NT – Polyaniline Nanotubes; ITO – Indium–Tin–Oxide; Ceo2 – Cerium Oxide; Sio2 – Sol-Gel Derived Silica; TB – Tributyrin; ERGO – Electrochemically Reduced Graphene Oxide; CHIT – Chitosan; Zno – Zinc Oxide; Znhcf – Zinc Hexacyanoferrate; Znonps – Zinc Oxide Nanoparticles; Irnps – Iridium Nanoparticles; NPG – Nanoporous Gold; Ptnps – Platinum Nanoparticles; PPY – Polypyrrole; FIA – Flow Injection Analysis.

Amperometric

Amperometric FIA

GPO/GK/AV membrane/Pt Enzymatic/FIA

LIP/GK/GPO/PtNPs/ PPY/ITO Enzymatic/FIA

ZnHCF/LIP/GK/GPO/ Au LIP/GK/GPO/CHIT/ ZnONPs LIP/GK/GPO/Inkjetprinted Au/PEDOT-PSS/ SPCE LIP/GK/GPO/MNPCHIT/ZnO-ZnHCF/GCE LIP/GK/GPO/MNPCHIT/ZnO-ZnHCF/GCE LIP/GK/GDH/IrNPs/ CPE LIP/NPG/GCE

Type of transducer

Amperometric FIA

Amperometric FIA

Amperometric

Amperometric

Amperometric

Amperometric

Amperometric

Amperometric

Amperometric

Type of biosensor

Table 1 (continued)

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Merits: ENPs have shown great promises in improving and simplifying the construction of enzyme electrodes due to their unique electronic, optical, mechanical, electrical, thermal and catalytic (ability to facilitate electron transfer) properties, beside the increasing the surface area of electrode and their direct attachment onto electrode through thiolate bond (Pundir et al., 2015). Table 1 summarizes the comparison of analytic characteristics of various triglycerides biosensors. This comparison shows that amperometric biosensors (working potential: − 0.7 to 1.2 V, pH: 6.0–11.0, response time: 2.5–1200 s, linearity: 0.002–100 mM, LOD: 10−7 mM to 14 mM) are better than potentiometric biosensors (working potential: − 0.02 to 0.2 V, pH: 6.0–7.4, response time: 30–2700 s, linearity: 0.024–30 mM, LOD: 9 × 10−5–5 mM) and DO metric biosensors (working potential: 0.4 V, pH: 6.5–7.8, response time: 2.0–300 s, linearity: 0.0005–20 mM, LOD: 0.0005–0.35 mM) 5. Summary and conclusion In conclusion, biosensing methods are comparatively superior than conventional colorimetric, enzymic colorimetric, spectrophotometric, chromatographic methods and fluorescence methods for determination of TG, as these are very simple, sensitive, selective, disposable, work with complete automation and provide rapid results. The TG biosensors work optimally within 2.5–2700 s at potential between − 0.02 and 1.2 V, pH range, 6.0–11.0, 25–9.5 °C and triolein concentration range, 0.001–100 mM with detection limit between 0.1 nM and 0.56 mM and can be reused upto 200 times over a period of 7–240 days, when stored at 4 °C. 6. Future perspective The TG biosensors are most promising device for clinical analysis of several metabolic disorders related to hypoxia and assessment of food quality. However, there is further need to develop more simple, accurate, reliable and cheap TG biosensors. The future research could be focused to design electronic chip and lab on paper chip to develop a fully automatic portable device, which can be used by the patients at his/her bedside. Labs on chip devices present many advantageous features such as minimal sample requirement, non-tedious and facile approach, cost-effective lab on a chip and fast response. The captivating features of paper based devices make it applied in determination of various serum metabolites (Narang et al., 2017). Conflict of interest Authors have no conflict of interest. Acknowledgement This work was supported by a grant (21(967)/13/EMR-II) from Council of Scientific and Industrial Research (CSIR), (New Delhi110012), to one of the author (CSP) as Emeritus Scientist. References Al-Hardan, N., Hamid, M.A., Ahmed, N., Jalar, A., Shamsudin, R., Othman, N., Keng, L.K., Chiu, W., Al-Rawi, H., 2016. High sensitivity pH sensor based on porous silicon (PSi) extended gate field-effect transistor. Sensors 16, 839. http://dx.doi.org/ 10.3390/s16060839. Aydemir, N., Malmström, J., Travas-Sejdic, J., 2016. Conducting polymer based electrochemical biosensors. Phys. Chem. Chem. Phys. 18, 8264–8277. http:// dx.doi.org/10.1039/c5cp06830d. Basu, I., Subramanian, R., Mathew, A., Kayastha, A.M., Chadha, A., Bhattacharya, E., 2005. Solid state potentiometric sensor for the estimation of tributyrin and urea. Sens. Actuators B: Chem. 107, 418–423. http://dx.doi.org/10.1016/ j.snb.2004.10.038. Batra, B., Narwal, V., Pundir, C.S., 2016. An amperometric lactate biosensor based on lactate dehdrogenase immobilized onto graphene oxide nanoparticles modified pencil graphite electrode. Eng. Life Sci. 16, 786–794.

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