20 Thin film biosensing application

Contents of this Unit

1.      Learning Outcomes

2.      Introduction

3.      Electrochemical Transducers

3.1    Three- electrode setup

3.2    Electrochemical transduction methods

4.      Matrix of a biosensor

4.1     Thin films as matrix

5.      Immobilization of biomolecules on a matrix

6.      Biosensing parameters

7.      Properties of a good biosensor

8.      Summary

1.      LEARNING OUTCOMES

The purpose of this module is to make students familiar with

• The different components of a biosensor
• Different methods of electrochemical transduction using a three- electrode setup
• Application of thin films in developing matrix for biosensors
• Various methods of immobilization of biomolecules on a matrix
• Biosensing parameters such as sensitivity, linearity, detection limit and Michaelis-Menten constant (Km)
• Properties that define a good biosensor

2.   INTRODUCTION

Biosensor is an analytical device that combines a biologically active component (receptor) with an appropriate physic- chemical transduction element (electrical, thermal, optical, etc.) for the detection of bioanalyte (biomolecules). The basic elements of a biosensor are as follows (figure 1):

1.      Receptor: It is a bio- molecular recognition element (for example: tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.) immobilized on the surface of a matrix. It reacts with a specific target analyte uniquely and selectively. For example, Gox for glucose.

2.      Matrix: It is a solid support for immobilization of the receptor. An ideal matrix ensures good stability even under adverse physiological conditions such as pH, temperature, etc., by maintaining the molecule’s bio- chemical properties and at the same time, exhibit good charge transfer characteristics. Hence, choice of a proper matrix is very crucial for the realization of an efficient biosensor.

3.      Transducer: It is the detector element (optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into a quantifiable electrical signal. Depending upon the type of signal, various transducers have been used in biosensors which work on different principles including electrochemical, optical, thermal, piezoelectric, etc. Amongst these, electrochemical transducers based on electron transfer are most widely used.

3. ELECTROCHEMICAL TRANSDUCERS

Electrochemical transducers sense a biochemical signal generated by a receptor via amperometric (measurement of change in current), potentiometric (measurement of change in potential) or conductometric (measurement of change in resistance) pathway and are widely used for biosensing applications. Electrochemical detection of biomolecules uses a three- electrode setup for various measurements.

3.1 Three – electrode setup

The different parts of a three- electrode setup are listed below (figure 2):

1. Working electrode: It is the microelectrode whose potential is varied with time with reference to another electrode (reference electrode).
2. Reference electrode: It is generally Saturated calomel electrode (SCE) or saturated Ag/ AgCl electrode whose potential remains constant.
3. Counter electrode: Mercury or Platinum are used as counters. It completes the circuit allowing conduction of electrons from signal source through the electrolyte to the working electrode.
4. Supporting electrolyte: An excess of nonreactive electrolyte (alkali metal) is required to conduct current.

3.2 Electrochemical transduction methods

Biosensors based on electrochemical transduction methods are explained below.

•   Conductometric biosensors: Conductometry is a technique depending on the change in conductivity in the solution due to the production or consumption of ionic species, for example, by the metabolic activity of the microorganisms. The measurement of conductance is extremely fast and sensitive under sophisticated modern analytical techniques, making conductometric biosensors very attractive. It is worth  noting  that  such  biosensors  are  suitable  for  miniaturization  since  it  requires  no  reference electrode in the system. However, all charge carriers could result in the change of conductivity, thus the selectivity of conductometric biosensors is relatively poor.

• Potentiometric biosensors: Potentiometry involves the measurement of the potential difference between the working electrode and the reference electrode and the potential signal exhibits concentration-dependent behavior. The transducer employed in the potentiometric technique is usually a gas-sensing electrode or an ion-selective electrode. The sensitivity and selectivity of potentiometric biosensor are outstanding due to the species-selective working electrode used in the system. However, a highly stable and accurate reference electrode is always required and which may potentially limit the application of potentiometric transducers.
• Amperometric biosensors: Amperometry involves the operation of the transducer at a given applied potential between the working electrode and the reference electrode and the current signal is recorded and correlated with the concentration of target compound. In the amperometric detection, the current signal is generated due to the reduction or oxidation of an electroactive metabolic product or intermediate on the surface of a working electrode. The resulting current is directly correlated to the bulk concentration of the electroactive species or its production or consumption rate within the adjacent biocatalytic layer. Due to the intrinsic sensitivity of current measurement, ultra-sensitive amperometric biosensors can be easily realized. The most direct approach for enzyme based biosensing is amperometric detection related to direct measurement of the oxidation (or reduction) current associated with the biochemical reaction.
• Impedimetric biosensors: Electrochemical impedance spectroscopy (EIS) is another important class of electrochemical biosensors which detects on the basis of change in impedance due to the occurrence of biorecognition event at the surface of bioelectrode. Impedance spectroscopy is a very powerful tool for the analysis if the interfacial properties of modified electrodes change on the occurrence of biorecognition events. EIS can be used to detect change in the impedance of the electrode-solution interface when the target analyte is captured by the probe. EIS is known to be one of the best techniques for fabrication of affinity based biosensors such as immunosensors.

4. MATRIX OF A BIOSENSOR

To maintain the bioactivity of biomolecules and to obtain the proper output signal, identification of an appropriate matrix for efficient immobilization of biomolecules is critical. The matrix should provide suitable conformation to the biomolecule for maximum activity and long-term stability. The identification and development of suitable matrices for the realization of biosensors exhibiting enhanced response characteristics such as stability, sensitivity, selectivity, reproducibility, shelf life etc. are crucial. These essential characteristics of biosensors demand to design desirable microenvironment for efficient immobilization of enzymes and to provide a fast electron transfer path between the enzyme’s active sites and the electrode.

Various materials have been exploited including metal oxides, self-assembled monolayers, metal nanoparticles conducting polymers and nanocomposites for development of matrices. The self-assembled monolayers and conducting polymers suffer from the specific problem of instability and degradation with time. However, metal oxides have profound applications as immobilization matrices as they not only possess high surface area, nontoxicity, good biocompatibility, high electro catalytic activity and chemical stability, but also shows fast electron communication features. Further, morphology of the matrix especially of nanostructured materials is one of the most important factors to determine the properties for biosensors since the biomolecules can be easily functionalized while they have very large surface area.

4.1 Thin Films as Matrix

Since the surface morphology of the thin films are easy to control by varying the deposition technique and deposition parameters in each technique, they are highly suitable as matrices in biosensors. Thin films based biosensors have an edge in terms of stability and reproducibility among other factors. A few examples of thin films based biosensors are listed in the table below.

5. IMMOBILIZATION OF BIOMOLECULES ON A MATRIX

Immobilization of a biomolecule is its confinement to a solid matrix or support resulting in reduction or complete loss of its mobility. Immobilization of receptors on the matrix surface is vital for developing efficient biosensors. In this process it must be ensured that the biomolecule retain its bio-specificity over time and over a certain range of parameters such as temperature and pH. In order to fully retain biological activity of the biomolecules after immobilization, biomolecules should be attached onto the surface of matrix without affecting its conformation and function. Several factors such as accuracy of measurements, sensor-to sensor reproducibility and operational lifetimes are drastically influenced by the enzyme stability. It is most important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemical nature or reactive groups in the binding site of the enzyme.

Methods of immobilization

There are four principal methods available for the immobilization of enzymes onto the matrix surface.

1.   Physical adsorption: The physical adsorption is the simplest approach but suffers with the problem of leaching out of the immobilized enzyme. Adsorption of the enzyme takes place on the matrix surface via weak forces such as ionic interactions, hydrogen bonds, van der Waals interactions, etc. Since most of the metal oxide matrices are positively charged and have high isoelectric point (IEP), they are most suitable for the immobilization of negatively charged enzymes having low IEP via electrostatic interactions.

2.   Covalent bonding: It is one of the most extensively researched techniques. In order to attach the enzyme, a covalent bond is formed between the chemical groups of enzyme and surface of matrix via functional groups. It leads to a very strong bond but the major shortcoming is that it may result in loss of activity and denaturation of immobilized enzyme due to the reactions at functional group sites.

3.   Entrapment: Enzymes are physically captured inside the matrix of a water soluble polymer and naturally derived gels e.g. agar, gelatin etc. However, there is a possibility of leakage of low molecular weight enzymes from the gel and thus, it does not find much applications.

4.  Encapsulation: Encapsulation is the enclosing of a droplet of solution of enzyme in a semipermeable membrane capsule. The method of encapsulation is less expensive and simple but there is a high diffusion barrier to both analyte and the product transport resulting in low activity.

6. BIOSENSING PARAMETERS

Careful design of a biosensor is crucial for medical applications as any false interpretation may lead to life threatening consequences. Thus, it is important to understand the various biosensing parameters such as sensitivity, linearity, detection limit and Michaelis-Menten constant (Km).

1.   Sensitivity: Sensitivity of a biosensor is an important parameter to evaluate its performance. It is defined as the change in magnitude of transduction signal in response to the change in analyte concentration. Sensitivity, S of amperometric sensor is determined by the slope of the calibration curve (i.e. graph between peak oxidation current [I] and concentration of analyte [S]) as

Nanostructured thin films have been proven to be highly sensitive due to large surface area provided by the nanostructures for the immobilization of biomolecules.

2.   Linearity: Linearity is defined as the constancy of the ratio of output (current) to input (analyte concentration). For a good biosensor, current, [I] is linearly proportional to the analyte concentration [S]. More is the linear range of biosensor, better is its response characteristic. A long linear range ensures detection of the analyte more accurately. This has been successfully achieved by thin films based biosensors.

3.   Michaelis- Menten constant (Km): Michaelis- Menten constant (Km) of an enzymatic reaction is a parameter that determines the affinity of immobilized enzyme towards its analyte. Lower value of Km indicates the high affinity of immobilized enzyme towards analyte. The value of Km is determined by plotting a graph, known as Hanes plot, between the analyte concentration ([S]) and analyte concentration/current ([S]/[I]). The ratio of slope of straight line of this plot to the intercept gives the value of Km.

4.   Detection limit (DL): Detection limit (DL) is another important parameter that can be deduced by considering noise in the transduction signal. If O is the minimum resolvable signal then detection limit is given by

DL = σ/S           …………………2

7. PROPERTIES OF A GOOD BIOSENSOR

A good biosensor is expected to fulfill some basic requirements as mentioned below:

1.   Accurate – A good biosensor should provide accurate and precise measurement over the useful range of analyte concentration.

2.   Sensitive – It should be able to detect even a small change in the measured variable (concentration of analyte).

3.  Specific – Its response should remain unaffected due to the presence of other interferants present in human biological fluids.

4.    Linear –Output signal should be linearly proportional to the value of measured variable (linear calibration curve)

5.  Simple – The designed biosensor should be simple in construction and economical.

6.  Reproducible – Response characteristics of biosensor should be reproducible.

7.  Stable – It should not degrade with time (Long shelf life).

8. SUMMARY

After studying this module, students should be familiar with

• The different components of a biosensor
• Different methods of electrochemical transduction using a three- electrode setup
• Application of thin films in developing matrix for biosensors
• Various methods of immobilization of biomolecules on a matrix
• Biosensing parameters such as sensitivity, linearity, detection limit and Michaelis-Menten constant (Km)
• Properties that define a good biosensor

 REFERENCES

1. Turner, Anthony; Wilson, George; Kaube, Isao (1987). Biosensors:Fundamentals and Applications. Oxford, UK: Oxford University Press. p. 770.
2. Bănică, Florinel-Gabriel (2012). Chemical Sensors and Biosensors:Fundamentals and Applications. Chichester, UK: John Wiley & Sons. p. 576.
3. A. Hierlemann, O. Brand, C. Hagleitner, H. Baltes, “Microfabrication techniques for chemical/biosensors, Proceedings of the IEEE 91 (6), 2003, 839–863.
4. Windmiller, Joshua Ray; Wang, Joseph (2013). “Wearable Electrochemical Sensors and Biosensors: A Review”. Electroanalysis. 25: 29.
5. Birkholz, Mario; Glogener, Paul; Glös, Franziska; Basmer, Thomas; Theuer, Lorenz (2016). “Continuously operating biosensor and its integration into a hermetically sealed medical implant”. micromachines. 7 (10): 183.
6. Kotanen, Christian N.; Gabriel Moussy, Francis; Carrara, Sandro; Guiseppi-Elie, Anthony (2012). “Implantable enzyme amperometric biosensors”. Biosensors and Bioelectronics. 35 (1): 14–26.