Which of the following is not considered as biomolecules?

6.5.1 Biosensors: Graphene-Based Electrochemical Biosensors for Detection of Biomolecules

Biomolecules play indispensable roles in all life processes, including disease development, so the accurate detection of biomolecules is critical to disease diagnosis and therapy. The graphene-based materials have been used to construct various biosensors based on different sensing mechanisms including optical and electrochemical signaling [101]. Because of its high sensitivity, low cost, quick response, and easy operation, the electrochemical approach is considered one of the best methods for biomolecule detection [38,102]. The excellent electrochemical properties of graphene make it a promising electrode material to improve the detection of biomolecules.

2.1 Biosensors

Biomolecules, range from small molecules, such as metabolites, to large molecules, such as protein and carbohydrates, which are chemical compounds produced by living organisms. These biomolecules are fundamental building blocks of living organisms, and therefore, the presence and appropriate concentrations of biomolecules are vital for the structure and proper function of living cells. Any changes in the concentration of specific biomolecules may lead to the malfunction of the cells and organisms. For this reason, the accurate measurement and monitoring of the concentration of specific biomolecules in a living system are crucial to ensure the well-being of the cells and living organism.

Biosensors are devices used for the detection and quantification of various biomolecules. Biosensors play important roles in our daily processes, especially in health diagnostics, biomedical engineering, pharmaceutical analysis, environmental analysis, food safety, and quality control and detection of toxic metabolites. Historically, several methods have been established for the recognition and determination of the concentration of biomolecules, such as flow injection analysis (Manuela Garrido et al., 1997), high performance liquid chromatography (Fotopoulou and Ioannou, 2002), spectrofluorimetry (Cañizares and Luque De Castro, 1995), spectrophotometry (Sorouraddin et al., 1998), flow-injection chemiluminescence (Michałowski and Hałaburda, 2001), capillary electrophoresis (Zhang et al., 2003), and electrochemical methods (Pingarrón et al., 2008). A typical biosensor contains two components, which are a biomolecule recognition element and a transducer. The recognition element is for biomolecular recognition, and generates a signal related to the concentration of the biomolecules. The biomolecule recognition element is specific to the biomolecule of interest, and therefore does not recognize biomolecules other than the analyte. These biomolecule recognition elements generally consist of biological probes made of cells or molecules such as aptamers, proteins, and nucleic acids. Additionally, enzymes that catalyze certain reactions involving electron exchanges can be used as biomolecule recognition elements. Transducers convert the signal from one form to another form. Transducers based on different mechanisms are developed and employed in biosensors, which can include mechanical, electrical, optical, electromagnetic, and electrochemical mechanisms (Zhang, 2015).

Introduction

Biomolecules such as DNA and proteins assemble into molecular machines and networks in biosystems such as cells. The functions performed in molecular machines and biosystems reflect the characteristic features of the biomolecules. Recent developments in molecular cell biology have allowed the biomolecules and their roles to be identified. Advances in structural biology have allowed the basic static structures to be determined with atomic resolution. These molecules, however, act in a kinetic and dynamic fashion, when they function. To understand the underlying mechanism of the functions it is therefore essential to monitor the dynamic and kinetic behaviors of biomolecules under their normal working conditions.

In conventional ensemble measurements faint signals from individual molecules are accumulated from a large number of molecules. In the averaging process the signals are amplified and the S/N ratio increased but some of the information such as the dynamic, kinetic, and fluctuating properties of biomolecules is hidden. Recent developments in lasers, detectors, and other techniques have made it possible to detect faint signals from individual molecules. These techniques have allowed the behavior of the individual biomolecules to be detected without averaging. The single-molecule measurements have provided unique information, which was not possible to obtain in the conventional ensemble measurements. The information is important particularly in elucidating the mechanisms underlying the function of biomolecules and biosystems.

Abstract

Biomolecules are an organic molecule that includes carbohydrates, protein, lipids, and nucleic acids. They are important for the survival of living cells. Some of valuable biomolecules have huge demand, which cannot be fulfilled from their renewable resources. Microbes have been used as a cell factory for their alternative production. The strategies adopted for engineering a high yielding biomolecule producing microbe are overexpression, knockout, and activation of gene. With the advent of synthetic biology and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas tool, genetic manipulation has become easier and is a less labor-intensive process. Engineering of metabolic pathway faces multiple challenges such as in metabolic flux imbalances, presence of nondesirable enzymes, and leaky gene expression can be solved by assembling the best biological parts available in nature. These biological parts can be used to construct a novel pathway for the production of the desired biomolecule without altering the native functions of the host. Another emerging area known as synthetic genomics has opened up possibilities of creating a synthetic genome for producing an artificial cell. Similarly, an artificial genome can be customized to engineer microbes for specific biomolecule production while removing nonessential function of the cell. This chapter describes the application of synthetic biology and CRISPR/Cas system for the production of important valuable biomolecules. The recent tools for the engineering of microbes are also discussed in the context of biomolecule production. The chapter overall produces a comprehensive work in the field of biomolecules production by applying diverse approaches of synthetic biology and CRISPR/Cas system.

4.3.4.1 General

Many biomolecules and microorganisms have been detected by means of nanomechanical sensors. Table 4.3.1 summarizes the reported studies [40]. Molecular recognition techniques play an important role in biosensing applications. Antigen–antibody interaction is one of the most powerful tools to detect biomolecules, making it possible to detect targets with high sensitivity and selectivity. Not only peptides including antigens and antibodies themselves, but also microorganisms such as viruses and fungi can be detected by antigen–antibody binding. Another promising technique to detect biomolecules is enzyme reactions. Enzyme reactions can be applied to detect smaller biomolecules such as hydrogen peroxide and glucose.

Table 4.3.1. Reported Studies of the Many Biomolecules and Microorganisms that have been Detected by Means of Nanomechanical Sensors [40]

Biomolecules

Biomolecules are all biological materials exclusive of cells and structural proteins when they are used as the “natural biomaterials” themselves. Biomolecules include proteins, lipids, etc., and can serve various functions like providing structural integrity to the tissue-engineered constructs. These include various growth factors, differentiation factors, and angiogenic factors essential in all categories of tissue engineering along with bone morphogenic proteins with a broad range of functional properties. Many biomolecules may assist the host with various functions like may support cell attachment, cell growth (or apoptosis), cell differentiation, cell migration, neovascularization, etc. All the functions indeed may be performed differently according to the biochemical, cellular, and biomechanical behavior.

Biosynthesis of Nanoparticles Using Extracted Biomolecules

Biomolecules are generally extracted by disrupting the algal cells obtained from their living cultures. In fact, this is the first ever reported method of alga-mediated NP synthesis process. Biomolecules from C. vulgaris were extracted to synthesize AuNPs [19]. To elaborate this method, biomass of C. vulgaris was first lyophilized and then subjected to reverse-phase high-performance liquid chromatography (RP-HPLC). This process yielded the gold shape-directing protein (GSP) that was responsible for producing gold nanoplates. In a different study, proteins of both low (PLW) and high molecular weights (PHW) from the biomass of the same algae were obtained by using a dialysis membrane [19]. However, only PHW was successful in promoting AgNPs.

Although extracted biomolecules have the same NP synthesis potential as living cultures, there are some fundamental differences between these two methods. Unlike the previous method, the current method requires some energy input owing to the implementation of some harvesting techniques. In general, different techniques can be applied to extract and produce biomolecules of various forms. Some of the commonly used techniques include vortexing with glass beads and sonication [69]. Therefore, the biomolecules obtained by applying these techniques can be in the forms of fine powder [70], cell-free filtrate of disrupted cells [71], cell-free supernatant [69], etc. However, the aspect of intra- and extracellular synthesis does not apply in any of these forms [19].

2 Electrochemical Sensing of Biomolecules Using Graphene–Metal Chalcogenide Composites

Biomolecules are important for the functioning of living organisms. Several macromolecules (protein, carbohydrates, nucleic acids, and enzymes) and small molecules (amino acids, vitamins, fatty acids, neurotransmitters, and hormones) fall under the category of biomolecules. These molecules perform or trigger important biochemical reactions in living organisms. When studying biomolecules, one can understand the physiological function that regulates the proper growth and development of a human body. Biomolecules may be either endogenous or exogenous depending on the involvement of biological process. The biomolecules may involve in several processes such as energy storage (carbohydrates), catalyzing the biochemical reactions (hormones), storing/transmitting the genetic codes (RNA/DNA), or altering biological and neurological activities (neurotransmitter/hormones). For proper growth and cell function, the dietary compounds are most important, because amino acids are the primary component of all dietary foods. When foods are consumed, they break down into the respective amino acids and transform into protein in the body. All the biological process require carbohydrates/amino acids to function properly and to regulate growth. Inadequate consumption of carbohydrates and amino acids will lead to various kinds of health ailments. Additionally, a low level of vitamin intake or improper secretion of hormones may lead to diseases; this is because they are involved in crucial physiological process such as growth regulation and in signaling pathways. For this reason it is highly important to monitor the biomolecules present in the human body.

Among various sensing strategies, the electrochemical sensing approach is more viable because of its portability, sensitivity, and selectivity compared to other methods. The development of ultrasensitive sensing of biomolecules is important from the perspective of early diagnosis and management of diseases [35,36]. In this section we will focus only on the electrochemical sensing of biomolecules (Cu ions, glucose, hydrogen peroxide, honokiol etc.) using graphene–metal chalcogenide composites.

One biologically-important metal ion is copper and its permissible limit is 0.8–0.9 mg day− 1 for normal adults. It is necessary for plants and animals as it maintains normal biological functions. However, it is also considered an important biological species that should be monitored, as its higher or lower concentration causes Wilson's disease, osteoporosis, kidney, liver damage, neutropenia, and infertility. According to the US Environmental Protection Agency (EPA), the amount of copper in drinking water must be less than 1.3 ppm. As a result, there has been ongoing interest in developing a sensitive and reliable Cu2 + ion sensor. The electrochemical sensing of Cu2 + ion is possible by the following redox reaction.

Cu2+⇌Cu++e−

Based on the redox behavior, graphene–SnS (Gr–SnS) nanocomposite modified glassy carbon electrode (GCE) as a Cu2 + sensor has been reported by Lu et al. [37]. They reported that Gr–SnS/GCE shows good sensitivity (detection limit of 0.02 μM) toward Cu2 + ion with linear concentration in the range of 1.5–36 μM. This sensor also exhibits good reproducibility and stability. On the other hand, Foo et al. [38] have reported the effect of visible light on electrochemical sensing of Cu2 + ions by using CdS/reduced graphene oxide on carbon cloth (CdS/rGO/CC) as a photoelectrochemical sensor. The linear range of this sensor is 0.1–1.0 μM and 1.0–40.0 μM with detection limit of 0.05 μM.

16.3.4 Biomolecule-based sensors

Biomolecule has two categories (Ni et al., 2017): macromolecules (e.g., protein, nucleic acid, polysaccharides) and micromolecules (e.g., amino acids, nucleic acids, monosaccharide) A biomolecule-based sensor has two types: enzyme based and DNA sensors. The DNA sensors are further classified as three major areas (Bishop, Satterwhite-Warden, Bist, Chen, & Rusling, 2016; Dirkzwager, Liang, & Tanner, 2016; Heger et al., 2016): (1) Fluidic device: This is introduced for ECL measurement and it has customizable/removable parts; (2) Aptamer device: Here malaria can be diagnosed using the principle of colorimetry. It has two prototypes; one is paper based and other is magnetic bead based; and (3) Stratospheric probe: It utilizes principle where DNA damage ca be evaluated (Bishop et al., 2016).

Introduction

Biomolecules such as enzymes, antibodies, affinity proteins, cell receptor ligands, and drugs of all kinds have been immobilized on and within biomaterial surfaces for a wide range of therapeutic, diagnostic, tissue regeneration, separation, and bioprocess applications. Immobilization of heparin on polymer surfaces is one of the earliest examples of a surface-modified, biologically functional biomaterial (Gott et al., 1963). Living cells may also be combined with biomaterials, especially when their surfaces contain cell adhesion peptides or proteins, and the fields of cell culture, artificial organs, and tissue engineering include important examples of cell–surface interactions. These “hybrid” combinations of natural and synthetic materials confer “biological functionality” to the synthetic biomaterial. Many sections and chapters in this textbook cover various aspects of this topic, including adsorption of proteins and adhesion of cells and bacteria on biomaterial surfaces, non-fouling surfaces, cell culture, tissue engineering, artificial organs, drug delivery, and others; this chapter will focus on the methodology involving physical adsorption and chemical immobilization of biomolecules on biomaterial surfaces, especially for applications requiring bioactivity of the immobilized biomolecule.

Among the different classes of biomaterials that could be biologically modified, synthetic polymers are especially interesting because their surfaces may contain reactive groups such as –OH, –COOH or NH2 groups, or they may be readily modified with other reactive groups such as azide, alkyne, and SH groups. All of these groups can be used to covalently link biomolecules.

Another advantage of polymers as supports for biomolecules is that the polymers may be fabricated in many forms, including films, membranes, tubes, fibers, fabrics, particles, capsules, and porous structures. Furthermore, macromolecular structures can also vary substantially. The latter can include homopolymers, random, alternating, block, and graft copolymers, hyperbranched (comb-like) and star-shaped structures (see Chapter I.2.2 on Polymers).

Living anionic polymerization techniques, along with newer methods of living free-radical polymerizations, now provide fine control of molecular weights with narrow distributions. The molecular forms of solid polymers include non-cross-linked chains that are insoluble at physiologic conditions, cross-linked networks, physical blends, and interpenetrating networks (IPNs) (e.g., Piskin and Hoffman, 1986; see also Chapter I.2.2). “Smart” polymers are sharply responsive in solubility behavior to stimuli, such as temperature, pH, and salt concentration (see Chapter I.2.11 on “Smart” Polymers).

For surfaces of metals, metal oxides, inorganic glasses or ceramics, biological functionality can sometimes be added via a chemically immobilized or physically adsorbed polymeric or surfactant adlayer, or by use of techniques such as plasma gas discharge, corona discharge in air or ozone to modify polymer surface compositions with functional groups (see also Chapter I.2.12). Several researchers have applied mussel adhesive chemistry based on self-condensation of dopamine to form tight bonding layers of polydopamine on a variety of surfaces, including metals, metal oxides, and glasses (Lee et al., 2007; Ku et al., 2010). The amine groups in these polymers may be further functionalized with biomolecules.