Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

Chapter

3

Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors CHAPTER OUTLINE

3.1.0 Introduction 105 3.2.0 Surface Functionalization

110

3.2.1 Modification of Metal Nanoparticles 110 3.2.2 Modification of Nanostructured Metal Oxides

113

3.3.0 Nanostructured Metals and Metal Oxides for Point-of-Care Diagnostics 3.4.0 Immunosensors Based on Nanostructured Metal Oxides and Metal Nanoparticles 121 3.5.0 Conclusions 122 References 122

116

3.1.0 INTRODUCTION The nanostructured metals and metal oxides have been predicted to have a great impact on biosensor and biomedical applications due to their unique chemical, electrochemical, and optical properties [1,2]. Nanostructured metals and metal oxides are known to be important materials due to their large surface area for immobilization of biomolecules with better orientation, conformation, and excellent bioactivity resulting in increased sensing performances. The performance of nanostructured metal and metal oxidesebased biosensors can be improved by tailoring the properties of the biointerface between nanomaterials and biomolecules via engineering of morphology, size, and functionality [3,4]. The noble metal nanoparticles (mNPs) including gold (Au), silver (Ag), platinum (Pt), palladium (Pd), nickel (Ni), etc. have been found to play a significant role toward the development of biosensors to achieve the demand of highly specific and highly sensitive diagnostics devices [4,5]. Several nanostructured metals have been employed for the fabrication of optical and electrochemical biosensors. The exciting properties of such Nanomaterials for Biosensors. http://dx.doi.org/10.1016/B978-0-323-44923-6.00003-0 Copyright © 2018 Elsevier Inc. All rights reserved.

105

106 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

nanoscale metals led to the development of a wide variety of chem- and biosensors including biosensors for point-of-care (POC) diagnostics devices, in vivo sensing, cell tracking, and therapy monitoring. The noble mNPs provide ease of functionalization with protein molecules via simple chemistry. The unique optical and spectral properties have stimulated the development of a plethora of sensing platforms. With the tuning feature of metal nanostructures by controlling morphological shape and size, the nanoparticles (NPs) can be implemented for specific applications in biosensing (Fig. 3.1.1) [6]. By varying chemical composition of the nano-scaled materials, their electrical, electrochemical, optical, and other exciting properties can be manipulated precisely [7]. The main features of a nanostructured metal are given below: n n n n

plasmonic excitation quantum confinement large surface-area-to-volume ratio large surface energies (local density of states)

n FIGURE 3.1.1 (A) Schematic representation of Kretschmann configuration for surface plasmon resonance. (B) Data are recorded as intensity variation of the reflected light at a fixed angle. (C) Corresponding sensorgrams for the specific interactions of the analyte with the spots on the surface [6].

3.1.0 Introduction 107

n n n n

short range ordering increased number of kinks large number of low-coordination sites with many “dangling bonds” ability to store excess electrons.

Many techniques have been employed to synthesize noble mNPs such as chemical and physical methods. Several chemical methods such as chemical and photochemical reduction, coprecipitation, thermal, hydrolysis etc. and physical methods like chemical/physical vapor deposition, laser ablation, grinding, etc. can be utilized to obtain NPs with better homogeneities, controlled sizes, shapes, and surfaces. The majority of mNPs are found to exhibit size-related properties compared with microparticles or bulk materials. In particular, Au NPs and Ag NPs are the mostly studied nanomaterials in the development of numerous techniques and tools for biosensors, imaging, drug delivery, and therapeutics [8]. Au NPs and Ag NPs are known to exhibit localized surface plasmon resonance (LSPR) properties that can be utilized for the development of new optical biosensors. When excited using light, the most noble mNPs are known to produce high absorption and scattering because of the collective oscillation of conduction electrons situated at the surface of the NPs. For Au NPs and Ag NPs, the LSPR provides high absorption coefficients and scattering within the range of ultravioletevisible wavelength which produces a higher sensitivity for optical detection systems than conventional organic dyes [9]. Au NPs have been widely studied because of their tunable optical properties and can be used in detecting, and imaging of biomolecules [10]. In addition to this, Au NPs are an important candidate for photothermal therapeutic, diagnostic, and drug delivery applications. Au NPs can enhance both Rayleigh and Raman signals to obtain chemical information on numerous biospecies of interest. Also, colloidal Au NPs produce strong vibrant colors due to surface plasmon resonance (SPR) absorption. SPR imaging for affinity-based biosensors have been described by Scarano et al. (Fig. 3.1.1) [6]. A fluorescence biosensor has been developed using selfassembled Au NPs probe for detection of hybridized DNA [11]. Ag NPs are widely used for the development of both optical and electrochemical biosensors. An LSPR-based optical sensor was demonstrated using triangular NPs of silver for affinity biosensor with high sensitivity [12]. By making nanocomposite of Ag NPs with other materials such as carbon nanotubes (CNTs) and with enhanced electrochemical property, the mediator-free biosensors can be developed. For example, a mediatorless hydrogen peroxide biosensor was developed using myoglobinimmobilized Ag NPseenabled CNTs film [13]. Other NPs such as Pt NPs

108 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

and Pd NPs have been employed to conjugate chitosan (Ch) and nafion, respectively, for development of electrochemical biosensors to determine total cholesterol and glucose in serum samples [14,15]. The nanostructured metal oxides (nMOs) have much interest as immobilizing materials for biosensors development [2]. The unique properties of nMOs offer exceptional prospects with interfacing bioreceptor molecules for biorecognition events with electronic signal transduction and for developing new generation of bioelectronics devices with novel functions [2]. nMOs can be categorized as inorganic, organic, and inorganiceorganic composites [16]. The inorganic nanostructures such as iron oxide (Fe2O3), zinc oxide (ZnO), nickel oxide (NiO), zirconium oxide (ZrO2), cerium oxide (CeO2), titanium dioxide (TiO2), silicon dioxide (SiO2), etc. have opened new opportunities because of their multifunctional properties [1]. Fig. 3.1.2 depicts the properties of some nMOs that can be used for the fabrication of biosensors. These nMOs can be utilized for the fabrication of electrochemical biosensors with excellent performances including high

ZrO , CH-Z

rO , C NT-Z

T-TiO

0 gO 12. M 8– es: on 9. r r tu ct P: a le r IE Fe t e sfe s n s: Fa tra et A rg N Ta G,D Ig

rO IEP: 4–11 Features: Bio compatible , binding with phosphorus ato m of DNA Targets: ChOx , GOx, lipase, HRP, IgG, DN A

, CN .2 -TiO , , CH : 3.9–8 patible O i P T IE iocom wide , B is : s s x, ture taly Fea hotoca andgap Ox, GO NA p b : Ch G, D ets P, Ig Targ se, HR lipa

SiO2 IEP: 1.7–3.0 Features: Biocompatible, functionality, morphology

NMOx Based Biosensors

Targets: ChOx,GOx, lipase, IgG, DNA

R

O

,C

H-

C

H

IEP:10.7 rp h adso r es: Hig Featur tron transfe elec

tion,

e,

x, lipas x, GO s: ChO NA IgG, D

Target

Ta r

Fe

at u

Ce

ts :C Ig hO G x , D ,G N Ox A ,

ge

Ta r

re s IEP eO ge ele : Bio : 6.7 , CN ts: ctr co –9. TCe H R Ch o n t mp 5 O P, Ox ran atib Ig ,GO sfe le, G, r f x a st DN , li A pas e,

Nio

P,

O Zn Tt N as ,C ,f le nO 5 b i Z t gy u- : 9. pa lo , A EP om n ho p nO I ioc tro or c -Z : B le om H es e nan ,C ur O t , r a Zn fe Fe ns tra

n FIGURE 3.1.2 Various nanostructured metal oxides (nMOx) for the fabrication of biosensors.

3.1.0 Introduction 109

sensitivity, cost-effectiveness, and a low limit of detection to quantify biomolecules. The electron transfer (ET) properties of nMOs are very significant to understand the carrier transport mechanism for electrochemical transducer fabrication. nMOs provide large surface-to-volume ratio, high surface reactivity, stability, chemical inertness, biocompatibility, and high electron transport properties [1,2]. However, these nMOs are problematic to functionalize that can perhaps be addressed by combining them with organic nanostructures, thus, in turn, a synergistic effect for designing new biosensor devices. nMOs can also increase the loading of biomolecules per unit mass of particles. The use of nMO permits for the possibility of numerous exciting technologies in biosensors arising from the nanoscale dimensions that can be utilized for rapid in vivo analysis. The morphological structure of nMOs such as shape and size can be tuned by various synthesis methods. The shape and size of nMOs can influence the properties of nMOs such as electron and phonon confinement, surface work function, surface reaction activity, high electro-activity, and adsorption ability. A variety of methods can be utilized to obtain different morphologies of nMOs such as soft templating for the preparation of NPs, nanorods, and nanofibers; solegels for the production of three-dimensional (3D) ordered nanostructures; radiofrequency sputtering; coprecipitation; chemical route; and hydrothermal with controlled shape. In electrochemical biosensors, bioreceptors coupled with nMOs-based electrode undergo redox reactions which can be electrochemically monitored [17]. A small mediator species during measurement can provide shuttles between the bioreceptors and the electrode. These nMOs can activate the bioreceptor (enzyme) active sites, and the electrochemical signal can be measured by direct ET. The active site of a biomolecule is found to be hidden deep inside the molecule structure. Thus, the direct ET can be difficult to achieve [18]. With the recent advances of nMOs, direct ET can be achieved by the modification of electrode surface using nMOs as mediators and by establishing functional biointerfaces (Fig. 3.1.3). Direct adsorption of biomolecules on the surface of bulk materials may cause denaturation, resulting in the loss of bioactivity. However, nMOs can be modified with carbon nanotubes and chitosan biopolymer to form nanocomposites and utilized them as biocompatible transducer materials as they retain the bioactivity of biomolecules such as enzyme, antibody etc. [19]. The formation of a biointerface between an nMO and a biomolecule is a key factor as the surface area, porosity, etc. can affect the efficacy of a biosensor. An effective biointerface can retain its bioactivity with better stability by introducing a biocompatible microenvironment with fast ET.

110 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

n FIGURE 3.1.3 Scheme of electron transfer (ET) process of nanostructured biointerfaces on the electrode surface [19]. nMOs, nanostructured metal oxides; Ox, oxidation.

3.2.0 SURFACE FUNCTIONALIZATION Surface functionalization of nanostructured metals and metal oxides continues to be a challenge for development of specific biosensors [20]. Different approaches have been explored for the functionalization of nanostructured materials by noncovalent and covalent interactions. Surface functionalization of a nanostructured material plays an important role toward stabilization, reduced aggregation, and high specific chemical reactivity toward other materials including biospecies and nanomaterials. In case of biosensor fabrication, surface functionalization of nanostructured materials can introduce various functional or organic groups and can also enhance hydrophilicity to attach biomolecules via establishing specific bonds or electrostatic interactions or by grafting of ligands on nanostructured materials. Surface functionalization can also stabilize mNPs against agglomeration, and modified mNPs are found to be compatible [21]. The mNPs can be made water soluble by attaching appropriate groups on their surface. Besides this, the surface modification may perhaps result in reduced homogeneity, compatibility with two phases, and enable self-organization of NPs.

3.2.1 Modification of Metal Nanoparticles By surface functionalization, the noble metals have been widely explored for different applications including catalysis, biochemical sensing, biolabeling, and photonics [22]. For example, Au NPs were exploited for the development of biosensors due to their good oxidation resistance and optical

3.2.0 Surface Functionalization 111

properties. Au NPs are good vehicles for tracers while Ag NPs are very good light absorbers and scatterers [20]. The surface of noble nanostructured metals can be modified by thiols [23], disulfides [24], amines [25], nitriles, carboxylic acids, and phosphine groups covalently [26]. Organosulfur compounds can be used for modification of the surface of mNPs due to the presence of organosulfur groups that can be directly conjugated to metal surfaces including Au, Ag, Pt, Pd, Cu, and Fe. It is known that sulfur (S) has a great affinity toward metal surfaces and also that metal-sulfur is strong enough to facilitate the immobilization of thiol groups on the surface of metals. The chemisorption energy of AueS is found to be 126 kJ/mol. Sulfur compounds can be grafted on the surface of mNPs wherein the solvent molecules are replaced by the sulfurcontaining ligands. Also sulfur can be directly attached during the synthesis of mNPs. Thiols can interact with mNPs by adsorption of the sulphydryl (RSH) groups on the surface of metals. And thiols or disulfides can be directly chemisorbed on metal surfaces which generates SR (R represents alkyl) groups, and chemisorption occurs by SeeH bond. However, the grafted organic molecules on the surface of mNPs may influence the properties of mNPs. The surface modification of mNPs by adsorbing amines can be performed to stabilize the NPs. For example, functionalization of Pd NPs by hexadecylamine provides good dispersion and better stability of the particles [27]. However, the interactions force between amino groups and mNP surfaces is found to weaker compared to thiolate groups. The amine-functionalized mNPs are generally bigger than organosulfur-modified NPs. Both methods can be applied for the modification of Ag NPs by peptides through thiol or amine functions. Noble mNPs can be stabilized by adsorption of tetraalkylammonium halides on the surface of NPs. For biosensor fabrication, a suitable immobilization method can be adopted for modification of selfassembled monolayer (SAM) on gold surfaces or Au NPs. Many immobilization chemistries can be performed after the formation of a thiolated layer. The thiol-modified gold surface can be directly attached to different biomolecules including DNA and RNA sequences (Fig. 3.1.4). Fig. 3.1.5 shows a schematic representation of colorimetric sensor for detection of adenosine using three different components such as gold NPs functionalized with 30 thiol-modified DNA (30 AdapAu), 50 -thiol-modified DNA (50 AdapAu), and a linker DNA (LinkerAdap) molecule [28]. The utilization of ligands for mNPs functionalization with more than one group may indicate multifunctional adsorption (Fig. 3.1.6). For example, a bifunctional material (N-isobutyryl-L-cysteine) on Au NPs via the thiol

112 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

n FIGURE 3.1.4 Various immobilization methods using chemical linkers. A self-assembled monolayer (SAM) is formed as the foundation of the array comprising alkanethiols-containing terminated amine, hydroxylic, or carboxylic functional groups. EDC, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; NHS, Nhydroxysuccinimide [6].

n FIGURE 3.1.5 Schematic representation of colorimetric detection of adenosine [28].

3.2.0 Surface Functionalization 113

n FIGURE 3.1.6 N-isobutyryl-L-cysteine (left); penicillamine (right) [20].

and carboxylate functions and a trifunctional material, penicillamine, with thiol, amine, and carboxylate interactions on surface may result in enhanced stability of the NPs. This functional property of the mNPs allows to make covalent amide bonds (CeN) with specific proteins or antibodies that can be used for the development of electrochemical and optical detection probes. Ali et al. have explained the multifunctionality of L-cysteine-capped quantum dots (Fig. 3.1.7) for attachment of Au substrate as well as antibodies to develop a low-density lipoprotein immunosensor [29]. The phosphines can also be utilized to functionalize mNPs. The Au NPs can be functionalized by triphenylphosphine [30]. The limitation of this method is that phosphine interaction with the mNPs is very weak resulting in their poor stability. However, the stability can be improved by the use of polyphosphine ligands. Pd NPs have recently been stabilized by using bis(diphenylphosphino) decane or bis(diphenylphosphinoethyl)phenylphosphine [31].

3.2.2 Modification of Nanostructured Metal Oxides There is an increasing demand to functionalize the surface of nMOs for the development of efficient biosensors. The properties of nMOs can be significantly altered by surface modification. Many organic compounds including thiols, carboxylic acids, and amines are explored to modify the surface of nMOs. However, modification of the nMOs with thiols and amines are rarely used. For example, thiourea can be utilized to modify tin oxide (SnO2) surface [32]. The majority of components used are phosphonates or silanes for modification of nMOs. Also, ligand exchange on the surface of nMOs can be performed without rearrangement of nMOs by having the same ligand charges by occupying the identical number of coordination sites. When a ligand is exchanged by an equally charged ligand (bi- or tridentate), the nMO surface requires rearrangement for accommodation of the additional ligand centers.

114 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

SH

CH2

COOH C

SH

SH EDC-NHS

Gold (Au) coated glass substrate

C

COOH COOH

NH2 CH2

NH2

NH2

Covalent bind formation

SH

L-cysteine capped CdS QD

C

CysCdS deposition on TGA/Au

CH2

SH

CdS

Thloglycolic Acid

COOH

COOH

SPR Response

EDC-NHS

SPR signal

Antibody (Apo-B100)

Impedance signal Impedance Response e–

OH CO

Detecter

LDL

COOH

CysCdS

LDL

LDL LDL Light Source

LDL LDL

Au AAR/CysCdS/Au electrode

V

LDL

Antigen-Antibody Complex

H COO

n FIGURE 3.1.7 Schematic representation of low-density lipoprotein immunosensor using in situ L-cysteine-capped cadmium sulfide quantum dots [29]. EDC, 1Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; LDL, Low-density lipoprotein; NHS: N-hydroxysuccinimide; QD, quantum dots; SPR, surface plasmon resonance; TGA, thioglycolic acid.

To modify the surface of nMOs, the carboxylate ligands such as fatty acids can be used. For example, carboxylic groups were recently utilized to bind with the surface of nanostructured titanium dioxide (TiO2) and investigated by a surface-binding transient probe molecule wherein the excited tripletstate probe molecule was produced by photoinduced interfacial charge recombination [33]. The carboxyl groups can also be used to obtain improved dispersion of iron oxide (Fe2O3) NPs in a biological system (wpH 7) by shifting the isoelectric point of the coated NP to wpH 3.0 [34]. In addition, silane moieties are widely used for the attachment of organic molecules by SieOeM bonding (Fig. 3.1.8). Several salines such as alkoxysilane, ^SieOR (R]alkyl); hydrogenosilane, ^SieH; or chlorosilane, ^SieCl, reagents etc. have been explored for modification of the nMOs. This method was applied to functionalize many metal oxide surfaces

3.2.0 Surface Functionalization 115

n FIGURE 3.1.8 Interactions of organosiloxane on the metal oxide surface.

such as SiO2, TiO2, ZrO2, SnO2, vanadium(V) oxide (V2O5), aluminium oxide (Al2O3), etc. With numerous functional abilities of salines (including amino, epoxies, cyano, carboxylic acid, etc.), they can be easily attached onto the surface of metal oxides. Silanes can also be used as a one-step method wherein it is introduced during the synthesis of metal oxides. Alkoxy- and chlorosilanes can interact with eOH groups on the surface of metal oxides by a condensation reaction. No water or catalyst is required in case of chlorosilanes (R2SiCl2 or RSiCl3) due to their high reactivity indicating that they can react with MeOH at the surface and adjacent MeOH groups leading to multidentate attachments or can form oligosiloxane structures via homocondensation reactions (Fig. 3.1.9). As a result, the structure of the organosiloxane layer may not be clearly defined. Thus, monochlorosilanes are mostly used in case of di- or tri-chlorosilanes. For nMOs surface modification, the organosilicon hydrides may have been considered important due to their clean reaction, byproducts being limited to hydrogen or water. These silanes have been successfully utilized for modification of ZnO, ZrO2, SiO2, and TiO2. Formation of SAMs of mercaptopropyltrimethoxysilane on ZnO has been explained by Petoral et al. [35]. The coupling agent such as 3-aminopropyltriethoxysilane can be used for modification of silicon oxide surfaces via surface hydroxylation of silicon and silicon dioxide or glass (Fig. 3.2.0). However, the surface functionalization may lead to more than one step of chemical treatment on biosensor resulting in device

116 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

n FIGURE 3.1.9 Mono-, bi-, or tridentate anchorage of a phosphonate ligand on a metal oxide surface [20].

OEt EtO NH

NH

OEt

Si

NH

NH

EtO

Si

EtO O

NH

NH

EtO

Si

Si O

O

O O O

O

Si O

O

Si O

O OH

Silicon n FIGURE 3.2.0 Hydroxylation of Si and SiO2 with 3:1 H2SO4:H2O2 via silanization reaction between (3-aminopropyl)triethoxysilane and the hydroxylated surface

[36].

complexity. Thus, efforts have been made to immobilize biomolecules on desired nMO surfaces via electrostatic interaction.

3.3.0 NANOSTRUCTURED METALS AND METAL OXIDES FOR POINT-OF-CARE DIAGNOSTICS The POC is considered important in clinical diagnostics and for the treatment of patients [37]. In this context, early diagnosis plays an important role in determining predisposition to a disease (prevention) or the outcome of disease (monitoring and prognosis). Electrochemical POC devices are generally small, portable, handheld, faster, and cost-effective devices [38].

3.3.0 Nanostructured Metals and Metal Oxides for Point-of-Care Diagnostics 117

These devices can be used for bedside, near-patient, satellite or remote, and decentralized testing. Electrochemical POC devices provide a convenient and immediate response to patients and the clinical care team. By utilizing electrochemical POC testing devices, results could be made available quickly to the clinical care team thereby facilitating immediate clinical decision and treatment device. Numerous tests including blood glucose, blood gas concentration, coagulation, expression of cardiac markers, drugs of abuse, pregnancy, hemoglobin (Hb), urine analysis, infected diseases, etc. could be feasible with POC devices. POC testing can be achieved via use of portable devices such as blood gluco/lactate meter, creatinine analyzer, C-reactive protein analyzer, pulse CO-oximeter, cholesterol meter, etc. With the goal of getting cheaper, smaller, faster, and smarter, the electrochemical POC devices have been considered to be one of the most favored technologies of the coming generation of health care diagnostic devices. nMOs and mNPs have recently captured increased interest for the development of electrochemical biosensors. The integration of nMOs and mNPs with electrochemical transducers is widely explored to realize compact POC devices. In a biosensor, the nMOs are known to play an important role for immobilization of biomolecules and direct ET between the electrode and electroanalyte. Incorporation of nMOs and mNPs on the electrode surface has been found to result in improved biosensor characteristics such as detection limit, sensitivity, selectivity, detection time, and long-term functional stability of the devices. NPs, nanocages, nanobelts, nanotubes, nanofibers, nanorods, etc. of metal oxides are widely used as transducer materials for development of electrochemical biosensors. nMOs have been used to catalyze biochemical reactions in the biosensor and have been predicted to exhibit novel functions. Consequently, the nMOs are being explored for application in drug delivery, tissue engineering, noninvasive sensors, in vitro and in vivo intracellular imaging, and biosensing. The common nMOs include Al2O3, SiO2, TiO2, ZrO2, ZnO, CeO2, MgO, Fe3O4, BaO, CuO, and V2O5 utilized in the development of biosensors. For example, titanium dioxide (TiO2) has attracted much interest in biomedical applications due to its high sensitivity, specificity, and fast detection of biomolecules [39]. These metal oxides offer high sensitivity due to their tunable band gap (1.8 and 4.1 eV) that depends on their crystal structures. The different crystalline structures can be obtained by tuning the temperature during annealing. TiO2 has four crystalline polymorphs namely rutile, anatase, brookite, and titanium dioxide (B). The size and shape of nanostructured TiO2 is predicted to play an important role in the construction of the biosensor. The high surface area of TiO2 NPs can be used to enhance biofunctionalization on the transducer surface [39]. The crystalline nature

Microfluidics system used for cholesterol detection

e-

118 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

Cholesterol Cholestenone

Flow direction Microchannel Inlet (200 µ) Connect to PDMS Syringe pump slab

Outlet with Ag/AgCI wire

Immobilization

ChOx

0

cm

3.0 cm

1.

Anatase - TiO2

Glass 1.2cm Substrate

Deposition of nanostuctured ant-TiO2

ITO

0.

2

cm

0.6 cm

Counter Electrode Working electrode (Bare ITO) (ChOx/ant-TiO2/ITO)

Biochemical reaction for ChOx\ant-TiO2/ITO electrode

n FIGURE 3.2.1 Pictorial representation of the microfluidic system for cholesterol detection [42]. ITO, indiuam tin oxide; PMDS, polydimethylsiloxane; PNA, peptide nucleic acid.

of nanostructured TiO2 can be used to improve the heterogeneous charge transfer between electrode and bulk solution. Viticoli et al. have discussed direct ET to construct a third-generation electrochemical biosensor using nanostructured TiO2 [40]. Cao et al. have developed a glucose biosensor using 3D macroporous TiO2 [41]. In addition, the nanostructured TiO2 provides optical transparency, biocompatibility, environmental safety, and excellent electrochemical conductivity resulting in improved biosensor characteristics. The good biocompatibility of TiO2 helps to decrease the enzyme inactivation rate. Ali et al. have described the construction of a microfluidic cholesterol biosensor using dispersed anatase TiO2 NPs [42] for blood cholesterol concentration monitoring (Fig. 3.2.1). Au, Ag, Pt, Cu, Pd, etc. have been widely exploited for the development of electrochemical-, as well as optical- or SPR-based biosensors. Because of easy surface functionalization to make nanocomposites with other nanomaterials and attaching biomolecules on the metal surfaces, Au NPs and Ag NPs have been found to exhibit excellent electrochemical properties for detection of various molecules. The functionalized mNPs with different biomolecules such as nucleic acids, antibodies, polymers, enzymes, and other proteins are found to increase biocompatibility and specificity of electrochemical biosensors. The dimensional similarities of mNPs with biological

3.3.0 Nanostructured Metals and Metal Oxides for Point-of-Care Diagnostics 119

n FIGURE 3.2.2 Schematic representation of 16s rRNA hybridization with peptide nucleic acid (PNA) probes and the signal amplification by an ionic interaction with gold (Au) nanoparticles [43].

species and their large surface areas can be used to obtain immobilized biomolecules with their bioactivities and conductivity to facilitate ET between the bioreceptor and electrode surface. The high stability and biocompatibility of mNPs allow them for easy conjugation with multiple biospecies, chemical groups, and polymeric materials. In addition, the unique electrocatalytic activity of mNPs such as Pt NPs could be employed to construct label-free electrochemical biosensors. To date, there are three approaches that have been developed for signal amplification in mNPs-based electrochemical biosensors. 1. mNPs are used as electroactive labels to amplify the signal. 2. mNPs act as carriers to load a large number of electroactive labels via covalent linkage or electrostatic interaction, which directly generate quantitative electrochemical signals. 3. Enzyme-conjugated mNPs can be exploited as labels to enhance the sensitivity through enzymatic catalysis. For example, a highly sensitive SPR sensor for detection of Escherichia coli 16s rRNA (16s rRNA is a genetic biomarker for recognition of organisms without analyzing polymerase chain reaction amplification because of its relatively high number of copies) was developed using peptide nucleic acid as a capture probe (Fig. 3.2.2) [43]. An electrochemical glucose sensor

120 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

SH

Au

SH

SH

SH dithiol

SH

gold nanoparticles

N H

2

SH

S

NH2 N H

2

S

S

NH2 H

2

cystamine

N

S

S

S

NH2 NH

S

2

S

NH2 NH2

=

O C

O=C H

H

GOx

C=O

C

H

=

H

O

S

N=CH

GOx

S

N=CH

GOx

S

N=CH

GOx

S

N=CH

GOx

IO4--Oxidized GOx

n FIGURE 3.2.3 Stepwise representation for functionalization of gold nanoparticles with dithiol, cystamine, and IO4  oxidized glucose oxidase (GOx) on a gold electrode for detection of glucose concentration [10].

has been fabricated [10] using functionalized Au NPs via dithiol and cystamine where glucose oxidase enzyme was covalently immobilized on the sensor surface (Fig. 3.2.3). Using functionalized Au NPs, a linear response to glucose the range of 2.0  105 to 5.7  103 M concentration, with a high sensitivity (8.8 mA/mM cm2) and a detection limit of 8.2 mM was observed for this glucose sensor [10].

3.4.0 Immunosensors Based on Nanostructured Metal Oxides and Metal Nanoparticles 121

3.4.0 IMMUNOSENSORS BASED ON NANOSTRUCTURED METAL OXIDES AND METAL NANOPARTICLES The immunoassays have been widely used for fast, selective, and sensitive detection of biomolecules [44]. With a similar working principle of the enzyme-linked immunosorbent assay (ELISA), most of the immunosensors are based on antigeneantibody interactions and formation on sandwich-like immunocomplex on the sensor surface. The sandwich-like immunocomplex consists of a specific antibody immobilized on the sensor surface, target antigen, and detection antibody for signal output. Immunoassays for the monitoring of tumor biomarkers have been employed for early cancer screening, diagnostics, and therapeutic applications. Several of the tumor biomarkers including carcinoembryonic antigen (CEA), alpha-fetoprotein (a-AFP), prostate-specific antigen, and interleukin-6 can be detected by nMOs- and mNPs-enabled transducers [7]. Specially, the mNPs play an important role in recognition of immunoreagents and amplify the signal transduction. Because of the amplification effect of mNPs, the mNP-based immunoassays have been found to be superior to ELISA in high detection sensitivity and selectivity. For example, nMPs-based electrochemical sensor shows a low limit of detection for a-AFP monitoring which is 107 magnitudes lower than commercially available ELISA kit (2.0  109 g/mL) [7]. Multiplexed tumor biomarker immunoassays have been found to be capable of detecting two or more species of tumor biomarkers simultaneously. They provide fast detection, improved detection efficiency, reduced costs, and decreased sample/reagent volume compared with single-analyte assays. However, multiplexed immunoassays need multiple signal readout with a specific identification for each species without overlaying each other. There are two strategies for multiplexed immunoassays such as spatially separated reaction zones and microarray systems. The nMOs and mNPs can be utilized for the development of immunosensors by modification of electrode surfaces. They provide a large surface area, rapid mass transport, facilitated ET, electrocatalysis, and biocompatibility. In particular, the biocompatibility of mNPs including Au NPs makes them suitable signal transducers and amplifiers for optical and electrochemical biosensors by carrying biospecies, electroactive tags, and redox complexes. An immunosensor was developed for detection of cardiac myoglobin in the blood plasma on the basis of the direct ET from the Fe(III)-heme to the electrode surface that was functionalized with mNPs and stabilized by didodecyldimethylammonium bromide and antibodies [45]. A multiplex immunosensor was based on antibody-conjugated nMO (iridium oxide) enabled by (3-aminopropyl)triethoxysilane and

122 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

n FIGURE 3.2.4 Schematic of an immunosensor consisting of two iridium oxide electrodes (W1 and W2) that functionalized with antibodies. 1 is a glass substrate of 20 mm  30 mm dimension, 2 is an electrical contact, 3 is an insulating layer of silicon carbide, and 4 is an acrylic well; sensing electrodes (W1 and W2) share a common external counter and reference electrode [46].

glutaraldehyde for detection of two different tumor biomarkers such as CEA and a-AFP simultaneously (Fig. 3.2.4) [46].

3.5.0 CONCLUSIONS In this chapter, we have described the application of nanostructured metals/metal oxides for the development of biosensors. With inherent benefits of nanostructured metals/metal oxides, these nanomaterials have been found to be of extensive use for the fabrication of biosensors to detect thousands of biomolecules including cancerous biomarkers and Alzheimer’s disease. The exciting properties of nanostructured metals/metal oxides, such as biocompatibility, large surface area, functional flexibility, and unique optical as well as electrochemical properties have been explored to enhance the selectivity and sensitivity of immunosensors. In next chapter, we discuss the utilization of biopolymeric nanostructures for biosensors and bioimaging.

REFERENCES [1] M.M. Rahman, A.J. Ahammad, J.H. Jin, S.J. Ahn, J.J. Lee, A comprehensive review of glucose biosensors based on nanostructured metal-oxides, Sensors 10 (2010) 4855e4886. [2] P.R. Solanki, A. Kaushik, V.V. Agrawal, B.D. Malhotra, Nanostructured metal oxide-based biosensors, NPG Asia Mater. 3 (2011) 17e24.

References 123

[3] F. Xiao, Y. Li, X. Zan, K. Liao, R. Xu, H. Duan, Growth of metalemetal oxide nanostructures on freestanding graphene paper for flexible biosensors, Adv. Funct. Mater. 22 (2012) 2487e2494. [4] A.J. Haes, S. Zou, G.C. Schatz, R.P. Van Duyne, A nanoscale optical biosensor: the long range distance dependence of the localized surface plasmon resonance of noble metal nanoparticles, J. Phys. Chem. B 108 (2004) 109e116. [5] G. Doria, J. Conde, B. Veigas, L. Giestas, C. Almeida, M. Assunção, J. Rosa, P.V. Baptista, Noble metal nanoparticles for biosensing applications, Sensors 12 (2012) 1657e1687. [6] S. Scarano, M. Mascini, A.P. Turner, M. Minunni, Surface plasmon resonance imaging for affinity-based biosensors, Biosens. Bioelectron. 25 (2010) 957e966. [7] J. Wang, Electrochemical biosensing based on noble metal nanoparticles, Microchim. Acta 177 (2012) 245e270. [8] K.S. Lee, M.A. El-Sayed, Gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition, J. Phys. Chem. B 110 (2006) 19220e19225. [9] C. Sönnichsen, B.M. Reinhard, J. Liphardt, A.P. Alivisatos, A molecular ruler based on plasmon coupling of single gold and silver nanoparticles, Nat. Biotechnol. 23 (2005) 741e745. [10] S. Zhang, N. Wang, H. Yu, Y. Niu, C. Sun, Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor, Bioelectrochemistry 67 (2005) 15e22. [11] D.J. Maxwell, J.R. Taylor, S. Nie, Self-assembled nanoparticle probes for recognition and detection of biomolecules, J. Am. Chem. Soc. 124 (2002) 9606e9612. [12] A.J. Haes, R.P. Van Duyne, A nanoscale optical biosensor: sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles, J. Am. Chem. Soc. 124 (2002) 10596e10604. [13] C.Y. Liu, J.M. Hu, Hydrogen peroxide biosensor based on the direct electrochemistry of myoglobin immobilized on silver nanoparticles doped carbon nanotubes film, Biosens. Bioelectron. 24 (2009) 2149e2154. [14] C. Nana, W. Hongjuan, L. Xiaomeng, Z. Liande, Amperometric glucose biosensor based on integration of glucose oxidase with palladium nanoparticles/reduced graphene oxide nanocomposite, Am. J. Anal. Chem. 3 (2012) 312e319. [15] M. Yang, Y. Yang, H. Yang, G. Shen, R. Yu, Layer-by-layer self-assembled multilayer films of carbon nanotubes and platinum nanoparticles with polyelectrolyte for the fabrication of biosensors, Biomaterials 27 (2006) 246e255. [16] A. Kaushik, R. Kumar, S.K. Arya, M. Nair, B.D. Malhotra, S. Bhansali, Organiceinorganic hybrid nanocomposite-based gas sensors for environmental monitoring, Chem. Rev. 115 (2015) 4571e4606. [17] A. Umar, Y.B. Hahn, Metal oxide nanostructures and their applications, Am. Sci. 5 (2010). [18] X. Lu, H. Zhang, Y. Ni, Q. Zhang, J. Chen, Porous nanosheet-based ZnO microspheres for the construction of direct electrochemical biosensors, Biosens. Bioelectron. 24 (2008) 93e98. [19] B.D. Malhotra, M. Das, P.R. Solanki, Opportunities in nano-structured metal oxides based biosensors, J. Phys. Conf. Ser. 358 (2012) 012007.

124 CHAPTER 3 Bioconjugated Nanostructured Metals and Metal Oxides for Biosensors

[20] M.A. Neouze, U. Schubert, Surface modification and functionalization of metal and metal oxide nanoparticles by organic ligands, Monatsh. für Chemie-Chem. Mon. 139 (2008) 183e195. [21] J. Virkutyte, R.S. Varma, Green synthesis of metal nanoparticles: biodegradable polymers and enzymes in stabilization and surface functionalization, Chem. Sci. 2 (2011) 837e846. [22] A.A. Lazarides, K.L. Kelly, T.R. Jensen, G.C. Schatz, Optical properties of metal nanoparticles and nanoparticle aggregates important in biosensors, J. Mol. StrucTheochem 529 (2000) 59e63. [23] R.C. Doty, T.R. Tshikhudo, M. Brust, D.G. Fernig, Extremely stable water-soluble Ag nanoparticles, Chem. Mater. 17 (2005) 4630e4635. [24] A. Ulman, Formation and structure of self-assembled monolayers, Chem. Rev. 96 (1996) 1533e1554. [25] M. Schulz-Dobrick, K.V. Sarathy, M. Jansen, Surfactant-free synthesis and functionalization of gold nanoparticles, J. Am. Chem. Soc. 127 (2005) 12816e12817. [26] E. Ramirez, S. Jansat, K. Philippot, P. Lecante, M. Gomez, A.M. Masdeu-Bulto, B. Chaudret, J. Organomet. Chem. 489 (2004) 4601. [27] C.I. Lynch, An overview of first-principles calculations of NMR parameters for paramagnetic materials, Mater. Sci. Tech. Ser. 32 (2016) 181e194. [28] J. Liu, Y. Lu, Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles, Angew. Chem. 118 (2006) 96e100. [29] Md A. Ali, S. Srivastava, M.K. Pandey, V.V. Agrawal, R. John, B.D. Malhotra, Proteineconjugated quantum dots interface: binding kinetics and label-free lipid detection, Anal. Chem. 86 (2014) 1710e1718. [30] G.H. Woehrle, J.E. Hutchison, Thiol-functionalized undecagold clusters by ligand exchange: synthesis, mechanism, and properties, Inorg. Chem. 44 (2005) 6149e6158. [31] E. Ramirez, S. Jansat, K. Philippot, P. Lecante, M. Gomez, A.M. Masdeu-Bultó, B. Chaudret, Influence of organic ligands on the stabilization of palladium nanoparticles, J. Organomet. Chem. 689 (2004) 4601e4610. [32] F. Liu, B. Quan, Z. Liu, L. Chen, Surface characterization study on SnO2 powder modified by thiourea, Mater. Chem. Phys. 93 (2005) 301e304. [33] Q.L. Zhang, L.C. Du, Y.X. Weng, L. Wang, H.Y. Chen, J.Q. Li, Particle-sizedependent distribution of carboxylate adsorption sites on TiO2 nanoparticle surfaces: insights into the surface modification of nanostructured TiO2 electrodes, J. Phys. Chem. B 108 (2004) 15077e15083. [34] S. Yu, G.M. Chow, Carboxyl group (eCO2H) functionalized ferrimagnetic iron oxide nanoparticles for potential bio-applications, J. Mater. Chem. 14 (2004) 2781e2786. [35] R.M. Petoral Jr., G.R. Yazdi, A.L. Spetz, R. Yakimova, K. Uvdal, Organosilanefunctionalized wide band gap semiconductor surfaces, Appl. Phys. Lett. 90 (2007) 223904. [36] R.G. Acres, A.V. Ellis, J. Alvino, C.E. Lenahan, D.A. Khodakov, G.F. Metha, G.C. Andersson, Molecular structure of 3-aminopropyltriethoxysilane layers formed on silanol-terminated silicon surfaces, J. Phys. Chem. C 116 (2012) 6289e6297.

References 125

[37] C.H. Ahn, J.-W. Choi, G. Beaucage, J.H. Nevin, J.-B. Lee, A. Puntambek, J.Y. Lee, Disposable smart lab on a chip for point-of-care clinical diagnostics, Proc. IEEE 92 (2004) 154e173. [38] H. Dong, C.-M. Li, Y.-F. Zhang, X.-D. Cao, Y. Gan, Screen-printed microfluidic device for electrochemical immunoassay, Lab. Chip 7 (2007) 1752e1758. [39] K. Mondal, Md A. Ali, V.V. Agrawal, B.D. Malhotra, A. Sharma, Highly sensitive biofunctionalized mesoporous electrospun TiO2 nanofiber based interface for biosensing, ACS Appl. Mater. Inter. 6 (2014) 2516e2527. [40] M. Viticoli, A. Curulli, A. Cusma, S. Kaciulis, S. Nunziante, L. Pandolfi, F. Valentini, G. Padeletti, Third-generation biosensors based on TiO2 nanostructured films, Mater. Sci. Eng. C 26 (2006) 947e951. [41] H. Cao, Y. Zhu, L. Tang, X. Yang, C. Li, A glucose biosensor based on immobilization of glucose oxidase into 3D macroporous TiO2, Electroanalysis 20 (2008) 2223e2228. [42] Md A. Ali, S. Srivastava, P.R. Solanki, V.V. Agrawal, R. John, B.D. Malhotra, Nanostructured anatase-titanium dioxide based platform for application to microfluidics cholesterol biosensor, Appl. Phys. Lett. 101 (2012) 084105. [43] H.A. Joung, N.R. Lee, S.K. Lee, J. Ahn, Y.B. Shin, H.S. Choi, C.S. Lee, S. Kim, M.G. Kim, High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor, Anal. Chim. Acta 630 (2008) 168e173. [44] L. Gervais, N. Rooij, E. Delamarche, Microfluidic chips for point-of-care immunodiagnostics, Adv. Mater. 23 (2011) H151eH176. [45] E.V. Suprun, A.L. Shilovskaya, A.V. Lisitsa, T.V. Bulko, V.V. Shumyantseva, A.I. Archakov, Electrochemical immunosensor based on metal nanoparticles for cardiac myoglobin detection in human blood plasma, Electroanalysis 23 (2011) 1051e1057. [46] M.S. Wilson, Electrochemical immunosensors for the simultaneous detection of two tumor markers, Anal. Chem. 77 (2005) 1496e1502.