Campus-XI, Patia Bhubaneswar-751024, Odisha, IndiaDepartment of Chemistry, Centurion University of Technology and Management, Odisha, India2
P.L. Nayak Research Foundation, Manorama Bhavan, Cuttack- 753004, Odisha, India3
Abstract: Biocompatible gold nanoparticles have gained considerable attention in recent years for potentialapplications in nanomedicine due to their interesting size dependent chemical, electronic and optical properties.In particular, the prospective use of gold nanoparticles as contrast enhancement agents in X-ray ComputedTomography (CT) and Photo Acoustic Tomography for early diagnosis of specific tumors is being extensivelyresearched. Additionally, gold nanoparticles show promise in enhancing the effectiveness of various targetedcancer treatments such as radiotherapy and photothermal therapy. For these applications, biocompatible goldnanoparticles labeled with specific tumor targeting biomolecules are needed for site specific delivery. Goldnanoparticles stabilized and labeled with carbohydrate (starch) and glycoprotein (gum arabic) have beengenerated, characterized and tested for in vitro and in vivo stability. They are found to localize in specifictissues in the animal models. Additionally, gold nanoparticles labeled with a cancer seeking peptide, bombesin,exhibited excellent binding affinity towards prostate and breast cancer cells. The degree of contrastenhancement in cancer imaging or effectiveness of cancer treatments is limited by the number of nanoparticlesthat can be localized at the target tumor/cancer site. The various biomedical applications of gold nano particleshave been discussed.
INTRODUCTION AuNP-based assays for clinical diagnostics which
Gold Nano Particle: Multifunctional nanoparticles, which capability and short turnaround times.incorporate diagnostic (quantum dots, magnetic, metallic, Gold nanoparticles represent a new class ofpolymeric and silica nanoparticles) and/or therapeutic biocompatible vectors capable of fulfilling this promise by(magnetic and metallic nanoparticles) properties, are in the selective cell and nuclear targeting of which will provideprocess of development. They have been used in vivo to new means for the site- specific diagnosis and treatmentprotect the drug entity in the systemic circulation, restrict of medical conditions. This work outlines theaccess of the drug to the chosen sites and to deliver the methodology for conjugation of AuNps with targetdrug at a controlled and sustained rate to the site of specific biomolecules and details theresults of studiesaction. The surface of gold nanoparticles can be tailored assessing the target specificity and cytotoxicity effects ofby ligand functionalization to selectively bind biomarkers. thus conjugated gold nanoparticles.Thiol-linking of DNA and chemical functionalization ofgold nanoparticles for specific protein/antibody binding Gold Nanoparticles and Their Properties: Goldare the most common approaches. Several methods have nanoparticles are defined as stable colloid solutions ofbeen utilized for detecting AuNPs such as scanometric, clusters of gold atoms with sizes ranging from 1-100 nmfluorescence, colorimetric, surface-enhanced Raman (Figure 1). At this nanoscale, AuNps possess differentscattering and electrochemical techniques. These unique physicochemical characteristics when compared to theaspects have allowed the development of novel bulk gold [1, 2], most obvious example being the color
promise increased sensitivity and specificity, multiplexing
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Fig. 1: Size of Gold nanoparticles number of reducing agents reported in the literature for
change from yellow to ruby red when bulk gold is Tri sodium citrate (Citrate synthesis) discovered byconverted into nanoparticulate gold. This ruby red color Turkevitch in 1973 [17] and sodium borate (Borateof AuNps is explained by a theory called “surface synthesis) introduced by Brust-Schiffrin in 1994 [18].plasmonics”. According to this theory, when the These two synthesis protocols pose potential problemsclusters of gold atoms are hit by the electromagnetic in case of size control, stability and most importantlyfield of the incoming light, the surface free electrons toxicity [19]. Therefore, we followed a novel protocol(6 electrons in case of AuNps) present in the conduction reported by Katti et al. in 2003 that uses aband of AuNps oscillate back and forth thus, creating a phosphino-amino acid based reducing agent, tris hydroxylplasmon band which has an absorption peak in the visible phosphine alanine (THPAL) to synthesize AuNps.region at 530-540 nm [3]. The surface plasmon band (SPB) THPAL is a water-soluble non-toxic reducing agent.of AuNps is used as an indicator for formation during It is reported that swine models can withstand up tothe synthesis of AuNps from their precursor salts. 100 mg/kg of body weight of THPAL with out showingThe sensitivity of plasmon band absorptivity is the basic toxicity, the most important of criteria in the use ofdetection mechanism involved in the AuNps based nanoparticles for bio medical applications. Due to thebio-sensors [4, 5]. Physical properties of AuNps in turn strong surface reactivity of free electrons present ondepend on the size, shape, particle-particle distance and AuNps, they easily tend to agglomerate posing stabilitythe nature of the stabilizer used to prevent the problems. Naturally occurring, FDA approved non-toxicagglomeration of nanoparticles [1]. According to Mie compounds such as starch; gum arabic and gelatin weretheory, Surface Plasmon Band (SPB) is absent for AuNps used to stabilize AuNps immediately after they are formedless than 2nm and greater than 500nm [2]. Gold nanorods [20]. These stabilizers form weak covalent bonds withhave two SPB’s, one longitudinalwavelength band at AuNps so that they easily shed off in the presence of550-600nm and one transverse-wavelength band at 520nm biomolecules with strong electronegative groups with[6, 7]. The longitudinal-wavelength band is very sensitive which the AuNps can then react.and changing the aspect ratio of Gold nanorods changesthe absorption region from visible to Near-infra red (NIR) Gold Nanoparticle-Based Targeted Delivery Systems:[8]. This unique optical property of Gold nanorods is used Gold nanoparticles; due to their ease of synthesis,in Near-infra red ray therapy [9]; and enhanced Raman uniform size distribution, rich surface chemistry and a lackscattering of adsorbed biomolecules [10]. Therefore, by of toxicity; have been proven to be excellent candidateschanging the size and shape of AuNps, the SPB and for conjugation with numerous biomolecules forscattering may be tuned for application in cellular imaging, site-specific delivery. Selective cell andn receptordrug delivery and therapy. The six free electrons present targeting of AuNps are likely to provide new pathways forin the conduction band of nanoparticulate gold makes the targeted delivery of diagnostic/ therapeutic agentsthem potential candidates to bind with thiols and amines [21, 22]. Tkachenko et al. showed specific nuclear[11]. Therefore, AuNps may be easily tagged with various targeting of AuNps by conjugating them with bovineproteins and bio-molecules rich in amino acids leading to serum albumin (BSA) using differential contrastimportant biomedical applications including targeted drug microscopy [23, 24]. Gold nanoparticles are used asdelivery [12, 13], cellular imaging [14] and biosensing [15]. targeted contrast.
Further, the free electrons also render AuNps useful ascontrast enhancement agents [16]. Imaging studies arebased on comparisons of contrast produced by thevariations in the electron densities in different tissues.With their high electron densities, AuNps serve asexcellent contrast enhancement agents in the detection oftumors.
Synthesis of Gold Nanoparticles: Gold Nanoparticles aretraditionally synthesized by reducing metallic gold in +3state to nanoparticulate gold in +1 state. There are a
the synthesis of AuNps. Two most important ones are:
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Approaches in Diagnostics Using AU-NP: improve the detection flexibility, simplicity and efficiency
Utilization of the AuNP color change upon that utilizes gold nanoparticles derivatized with thiolaggregation, the best characterized example being modified oligonucleotides was developed recently.AuPs functionalized with ssDNA capable of The gold nanoparticles probes, GP /GP-2 for IS6110 andspecifically hybridizing to a complementary target for GP-3/GP-4 for Rv3618, were designed to specificallythe detection of specific nucleic acid sequences in hybridize with target DNAs of MTBC and MTB strains,biological samples [15]. respectively [20]. Use of AuNPs as a core/seed that can be tailoredwith a wide variety of surface functionalities to Specific DNA and RNA Detection Based on AuNPs:provide highly selective nanoprobes for diagnosis DNA and RNA carry informatins and is very interesting[16]. molecule in diagnostics and clinical research. To start withUtilization of AuNPs in electrochemical based diagnosis first step is to identify the conserve genemethods that can be coupled with metal deposition sequences.. Many workers have identified differentfor signal enhancement [17]. ligand molecules associated with different disease
AU NPs-Based Clinical Diagnostic Technology nanoparticles (herein designated Au-nanoprobes) for theNanoparticle in Cancer Targeting: Nanoparticles with colorimetric detection of gene targets represents anunique optical properties, facile surface chemistry and inexpensive and easy to perform alternative toappropriate size scale are generating much eagerness in fluorescence or radioactivitybased assays [24]. In 1996,clinical diagnostics. Gold nanoparticles have immense Mirkin et al. [25] described the use of single-strandedpotential for cancer diagnosis and therapy on account of oligonucleotide targets that could be detected usingtheir surface plasmon resonance (SPR) enhanced light two different Au-nanoprobes such that each wasscattering and absorption. Conjugation of Au functionalized with a DNA-oligonucleotidenanoparticles to ligands specifically targeted to complementary to one half of the given target.biomarkers on cancer cells allows molecular-specific Mehmet Ozsoz et al reported electrochemical genosensorsimaging and detection of cancer. Additionally, Au for the detection of the Factor V Leiden mutation fromnanoparticles efficiently convert the strongly absorbed polymerase chain reaction (PCR) amplicons using thelight into localized heat, which can be exploited for the oxidation signal of colloidal gold (Au) nanoparticle [26].selective laser photothermal therapy of cancer has been In this study a pencil graphite electrode (PGE) modifieddscussed. Recently use of selectively targeted Au with target DNA, when hybridized with complementarynanospheres in cancer photodiagnostics and probes conjugated to Au nanoparticles, responded withphotothermal therapy have been developed [18]. the appearance of a Au oxide wave at +1.20 V. SpecificIn another report potential use of nucleic acid ligand probes were immobilized onto the Au nanoparticles in two(aptamers) conjugated gold nanoparticles (AuNPs) for different modes. The detection limit for the PCR ampliconscancer cell detection was explored. This was due to was found to be as low as 0.78 fmol; thus, it is suitable forspecific binding of the aptamers toward platelet-derived point-of-care application. Recently Li Wang et al reportedgrowth factor (PDGF), MDA-MB-231 and Hs578T cells piezoelectric genosenosr (QCM) biosensor for real-time(cancer cells) that over-express PDGF, interact with detection of E. coli O157:H7 DNA based on nanogoldApt-AuNPs to a greater e xtent than do particles amplification [27]. This paper presentsH184B5F5/M10 cells (normal cells). This results were development of a quartz crystal microbalance (QCM)confirmed through inductively coupled plasma mass biosensor for real-time detection of E. coli O157:H7 DNAspectrometry measurements of the gold ion based on nanogold particles amplification. Many inner Auconcentrations within these cells [19]. nanoparticles were immobilized onto the thioled surface of
Nanoparticle Based Diagnosis of Tuberculosis: stranded DNA (ssDNA) probes could be fixed throughTuberculosis (TB) is a common and often deadly Au-SH bonding. The hybridization was induced byinfectious disease caused by mycobacteria, usually exposing the ssDNA probe to the complementary targetMycobacterium tuberculosis in humans. Many methods DNA of E. coli O157:H7 gene eaeA, then resulted in aso far developed for diagnosis of tuberculosis. To further mass change and corresponding frequency shifts ( f) of
and reduce the cost, advance molecular diagnosis assay
1
[21-23]. The use of thiol-linked ssDNA-modified gold
the Au electrode, then more specific thiolated single-
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the QCM. The outer avidin-coated Au nanoparticles diagnostics and therapeutics by merging thecould combine with the target DNA to increase the mass. characteristic properties they possess at the nanometricThe electrochemical techniques, cyclic voltammetry (CV) scale with the feasible immobilisation of specific ligandsand electrochemical impedance spectroscopy (EIS) on the surface. Therefore, they have become idealwere adopted to manifest and character each step. candidates for molecularly sensitive detection, highlyThus nanoparticle associated with the biosensing system efficient contrast agents for molecular imaging, as well ascan be use for various applications, to produce diagnostic carriers for targeted drug and gene delivery andtools. therapeutical reagents for targeted photothermal therapy.
Potential Therapeutic Application of Gold Nanoparticles: behavior of nanomaterials in biological systems needs toIn medical term a therapeutic effect is a consequence of a be addressed, as well as the engineering of novelmedical treatment of any kind, the results of which are nanoparticles, which can overcome the drawbacks relatedjudged to be desirable and beneficial. Due to advances in to currently developed nanomaterials, includingnanobitechnology, potential therapeutic application of nonspecific binding, aggregation, toxicity andgold nanoparticles has been investigated by different biodistribution.coworkers. P Mukherjee et al studied B-Chronic Biocompatible gold nanoparticles have gainedLymphocytic Leukemia (CLL) which is characterized by considerable attention in recent years for potentialapoptosis resistance [28]. They found induction of applications in nanomedicine due to their interesting sizesignificantly more apoptosis in CLL B cells by co-culture dependent chemical, electronic and optical properties.with an anti-VEGF antibody. To increase the efficacy of In particular, the prospective use of gold nanoparticles asthese agents in CLL therapy they focused on the use of contrast enhancement agents in X-ray Computedgold nanoparticles (GNP). By attaching VEGF antibody Tomography (CT) and Photo Acoustic Tomography for(AbVF) to the gold nanoparticles they determined the early diagnosis of specific tumors is being extensivelyability to kill CLL B cells. All the patient samples studied researched. Additionally, gold nanoparticles show(N = 7) responded to the gold-AbVF treatment with a dose promise in enhancing the effectiveness of variousdependent apoptosis of CLL B cells. The induction of targeted cancer treatments such as radiotherapy andapoptosis with gold-AbVF was significantly higher photothermal therapy. For these applications,than the CLL cells exposed to only AbVF or GNP. biocompatible gold nanoparticles labeled with specificThe gold-AbVF treated cells showed significant down tumor targeting biomolecules are needed for site specificregulation of anti-apoptotic proteins and exhibited PARP delivery. In the present project, gold nanoparticlescleavage. Gold-AbVF treated and GNP treated cells stabilized and labeled with carbohydrate (starch) andshowed internalization of the nanoparticles in early and glycoprotein (gum arabic) have been generated,late endosomes and in multivesicular bodies. Non-coated characterized and tested for in vitro and in vivo stability.gold nanoparticles alone were able to induce some levels They are found to localize in specific tissues in the animalof apoptosis in CLL B cells. Thus this paper opens up models. Additionally, gold nanoparticles labeled with anew opportunities in the treatment of CLL-B using gold cancer seeking peptide, bombesin, exhibited excellentnanoparticles and integrates nanoscience with therapy in binding affinity towards prostate and breast cancer cells.CLL. The degree of contrast enhancement in cancer imaging or
Gold-based nanoparticles such fascinating features effectiveness of cancer treatments is limited by theas ease of synthesis and surface functionalization with number of nanoparticles that can be localized at the targetthiol-containing molecules, non-cytotoxicity, high tumor/cancer site. One way to augment the localization ofbiocompatibility, as well as broad-based optical nanoparticles at the target tissue is to utilize goldproperties, make gold-based nanoparticles still another nanochains that hold more number of nanoparticles.attractive nanomaterial and one of the most studied in the Therefore, we developed biocompatible gold nanochainsbioanalytical field. Low nonspecific binding in control formed by self assembly of nanoparticles on gum arabiccells. Nanobiotechnology has become an attractive and and were shown to be in vitro stable.promising research area with potential application in many The change of optical properties of golddiversified fields and it has played a particularly important nanoparticles upon slight modification of the surroundingrole in clinical research and biomedicine. Nanoparticles environment is the basis for the development ofhave emerged as promising nanoplatforms for efficient biosensors. Therefore, Surface Enhanced Raman
Nonetheless, a better fundamental understanding of the
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Scattering (SERS), a spectroscopic method where the number of ~ 100 nm pores. Additionally, theseRaman scattering signal, which is sensitive to the discontinuous capillary walls have no basal laminamolecular structure,is enhanced in the presence of gold allowing particles less than 100 nm to penetrate easilynanoparticles has emerged as a powerful tool for the [28]. Interestingly, tumor tissues lack a lymphatic systemdetection of specific molecules. Consequently, there is for eliminating lipophilic and polymeric materials fromneed for nanostructures that give maximum SERS signal. them [29]; therefore, once the particles penetrate theIn the present project, gold nanoparticles set in agarose tumor tissues, they cannot be eliminated easily.gel have been demonstrated to be excellent SERS Accordingly, tumors exhibit enhanced penetration andsubstrates compared to commercially available gold retention effect (EPR effect) for 50-100 nm particles.nanoparticles for DNA nucleosides. AuNPs conjugated to appropriate tumor avid
Bulk gold is well known for being inert; however, the biomolecules with mean sizes in50-100 nm range are idealnanoparticulate sizes of gold display astronomically high for targeting tumors for imaging and therapy purposes.chemical reactivity [16, 17]. Consequently, the rich surface Connor et al. have shown that gold nanoparticles arechemistry of AuNPs allows surface modification reactions inherently non-toxic to human cells. despite being takenwith wide varieties of chemical and biochemical vectors to up into the cells. However some precursors used totailor to the needs of biomedical applications including generate nanoparticles might be toxic [30]. This result isimaging and therapy of cancer [18-20]. Hybrid AuNPs are significant for the toxicity of gold nanoparticles can beproduced by the interaction of highly reactive nascent controlled by using non-toxic reagents to produce them.AuNPs with chemical functionalities present on simple Optical and electronic properties of AuNPs can bechemical molecules or on specific molecules of biological utilized to enhance the contrast in molecular imaging forinterest (for example biomolecules including peptides and the detection of cancer at early stages. For example,proteins) [13, 21]. These hybrid AuNPs labeled with tumor AuNPs labeled with monoclonal antibodies against EGFRseeking biomolecules provide efficient vehicles for site (epidermal growth factor receptor) that are over-expressedspecific delivery of nanoparticles carrying imaging and in skin cancer were shown to localize on the abnormaltherapeutic probes to target cancer cells [22-25]. In X-ray cervical SiHa cell lines by imaging via endoscope-CT imaging, the contrast appears due to variations in the compatible microscopies, such as optical coherenceelectron densities (in turn attenuation coefficients) of tomography and reflectance confocal microscopy [31, 32].different tissues. Gold being a metal with high electron The AuNPs appeared as bright spots on the surface ofdensity, tumor site specific hybrid AuNPs can be used as cell lines in a bright field image. Hainfeld et al. haveeffective contrast agents in X ray CT imaging [26].Gold demonstrated the use of gold nanoparticles as X-raynanoparticle solutions are bright red/purple colored due contrast agents in imaging breast cancer. 1.9 nm AuNPsto plasmonabsorption. Any surface modification of were injected via a tail vein into Balb/C mice bearingAuNPs results in the shift of plasmon EMT- 6 subcutaneous mammary tumors and imaged by aabsorptionwavelength. This change in optical property of clinical mammographic unit. A 5 mm tumor growing in oneAuNPs when coming in contact with the probe thigh was clearly evident from its increased vascularitybiomolecule is exploited to develop biosensors [13, 27]. and resultant higher gold content in the X-ray image [26].
Gold Nanoparticles in Cancer Diagnosis and Therapy: chemotherapy, photo-thermal therapy and radiotherapy.It is important to understand the difference between AuNPs have been investigated for potential candidates tonormal and canceroustissue to effectively develop hybrid assist in photo-thermal therapy and radiotherapy. O’Nealnanoparticles in cancer diagnosis and therapy.Normal et al. examined the feasibility of nanoshell-assistedtissues have tight, continuous vessel walls interspersed photo-thermal therapy (NAPT). Polyethylene glycol (PEG)with 9 nm poresfrequently and 50 nm pores infrequently. coated nanoshells, (<130 nm diameter) consisting of silicaTherefore, small molecules can easily penetrateall types of core with a gold shell and exhibiting an absorption peaktissues in contrast to large molecules such as polymers in the 805-810 nm region, were injected intravenously intothat do so very slowly.However, tumor tissues, a mice bearing subcutaneously grown colon tumor.inflammatory tissues and reticuloendothelial system The nanoshells accumulate at the tumor site due to(RES)-rich organs, such as the liver, spleen and bone enhanced penetration and retention effect (EPR effect)marrow have discontinuous capillary walls and a large over a 6 hr period.
Treatment of cancer has different routes such as
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Subsequently, the tumors were exposed to NIR light(808 nm diode laser, 800 mW) resulting in absorption ofinfra-red light by nanoshells and consequent generationof heat that caused irreversible damage to the tumor.In this study, the nanoshell-assisted photothermaltreatedtumor displayed complete regression and these miceremained healthyand tumor-free for < 90 days followingtreatment, unlike the sham group (exposure toNIR alone)and the control group (no injection and exposure) wherethe tumors keptgrowing [33]. This outcome providesimpetus to develop AuNPs with NIR absorbanceforeffective treatment of cancer. Hainfeld et al. demonstratedthat the irradiation of AuNPs accumulated in tumor with250 kVp X-rays caused shrinkage of tumor in mice with Fig. 2: General strategy for on chip biosensorsubcutaneously grown mammary carcinoma tumor. It wasalso found that that treatment with X-rays alone had no In addition to the above mentioned principle for designingtherapeutic effect on the tumor [34]. This study illustrates biosensors, Surface Enhanced Raman Scattering (SERS)the possible use of AuNPs in radiotherapy with use of has emerged as a powerful spectroscopic tool that [40]X-rays. These examples of AuNPs in cancer therapy can be employed in detecting trace amounts of moleculesdemonstrate the potential of AuNPs in therapy but are yet adsorbed on or present near metallic nanostructures alongto comprise target specificity by their conjugation to with structural and molecular information of thesuitable biomolecules.Recent work by Kannan and Katti molecules.SERS now provides a great potential for label-et al. investigated gum arabic labeled radioactive AuNPs free detection of biomolecules [41, 42]. Significant effortsthat localize in liver. This study combines the have focused on the binding of the oligonucleosides totherapeutic property of radioactive gold 198Au metal surfaces and colloids for a variety of applications,(_max = 0.96 MeV, t1/2 = 2.7 days) and target specific including multiplexed DNA detection technology [43],biomolecule to form a powerful radiopharmaceutical for rapid sequencers based on SERS from single DNA basestargeted drug delivery. This gum Arabic labeled [41] and real-time DNA detection methodology. For all theradioactive gold can be used to treat liver cancers with above applications new strategies for the synthesis,higher radiation dose inherent to radioactive DNA-nanoparticle assembly methods and development ofnanoparticles that contain thousands of radioactive atoms nanoparticle-based SERS-active substrates are needed.[35]. In the present project we have developed a new SERS
Gold Nanoparticles in Biosensor Applications: The basic that provides better enhancement in Raman signal ofprinciple involved in the design of a biosensor based on DNA nucleosides than that with commercially availablegold nanoparticles is that the AuNPs are functionalized or gold nanoparticles. Before moving on to the projectcapped with a thiolated biomolecule which upon outline the following section discusses Raman scatteringidentifying the complementary biomolecule causes and SERS.change in the optical absorption of AuNPs [36].For example, aptamer functionalized AuNPs specifically Bioconjugation of AuNPs : Hybrid gold nanoparticles arebinds to thrombin causing aggregation of AuNPs and red produced by the interaction of highly reactive nascentshifting the plasmon peak. The specific binding was AuNPs with chemical functionalities present on specifictested by exposing aptamer functionalized AuNPs to molecules of biological interest (including peptides andother proteins (BSA or human IgG antibodies) where no proteins). The conjugation protocols that are applied forAuNP aggregation was observed [37]. Similarly, production of radiolabelled bioconjugates, traditionallyimmunoassays have been based on antigen-antibody used for cancer diagnosis and therapy [44-47], can beinteractions. AuNPs functionalized with antigen extended for the labeling nanoparticles of gold and other(antibody) aggregate when matching antibody (antigen) metals with tumor specific peptides. A hybrid goldbinds causing shift in the plasmon absorption [38, 39]. nanoparticle has 4 components: (i) AuNP, (ii) Chelating
substrate based on gold nanoparticles in agarose matrix
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Fig. 3: Design strategy for bioconjugate /hybrid gold considerable attention because they are easy andnanoparticles economical to mass production, they are robust and they
Fig. 4: Dsplacement of starch with sulphur containing novel and sensitive electrochemical immunoassay formoieties such as thiols in starch stabilizied gold immunoglobulin G (IgG) has been developed by Limogesnanoparticles and co-workers using a colloidal gold label via anodic
moiety, (iii) Linker/Spacer and (iv) Cancer seeking peptide (concentration as low as 3×10-12 M) could be obtained,In the present project, starch stabilized AuNPs are which was competitive with colorimetric enzyme linkedutilized. The nature of bonding between starch and immuno-sorbent assay or with immunoassays based onAuNPs is a weak coordination bond between the hydroxyl fluorescent europium chelate labels. Furthermore, Shen’sgroups in starch and gold. However in presence of group reported a novel electrochemical immunoassaypowerful electron donor atoms such as S, this weak based on the precipitation of silver on colloidal goldcoordination bond is expected to break and starch labels. After metal silver dissolution in an acidic solution,molecules detach from gold (Figure 1.9). The biomolecule the signal was indirectly determined by anodic strippingchosen for bioconjugation with AuNPs is the voltammetry at a glassy carbon electrode.A detectionseven-amino acid truncated bombesin analogue limit as lowas 1 ng mL human IgG was achieved.(BBN8-14) that is known to target gastrin releasing The enhancement in sensitivity for an electrochemicalpeptide (GRP) receptors that are over expressed on in a immunoassay by the autocatalytic deposition ofvariety of neoplasma including small cell lung, prostate, Au3+ onto AuNPs has been studied by Huang’s group.breast, gastric, pancreatic, gastrointestinal carcinoid and By coupling the a utocatalytic deposition withcolon cancers[48-56]. To impart specificity hybrid AuNP, square-wave stripping voltammetry, the rabbitdisulfide moiety is chosen as a chelating moiety. S-S immunoglobulin G analyte could be determinedgroup undergo oxidative addition to AuNP and the quantitatively. A very low detection limit, 0.25 pgmLreaction is very selective, even in the presence of thiol (1.6 fM) was obtained, which is three orders of magnitudegroups. Thioctic acid, a biological antioxidant [57] and lower than that obtained by a conventional immunoassaybelieved to exhibit metal chelating properties [58], using the same AuNPs labels. Novel enzyme-labeledcontains disulfide and carboxylic acid groups to electrochemical immunosensors were well developed byconjugate to peptide. S-S group acts as a chelating moiety several groups. For instance, Ju’s group reported that ato hold the AuNP and 5 carbon atoms act as a spacer highly hydrophilic and conductive colloidalbetween S-S and the biomolecule. The thioctic acid AuNPs/titania sol–gel composite membrane could bemodified bombesin is used for AuNP bioconjugation. employed as electrochemical sensing interface for
Application of Gold Nanoparticles for Immunosensors: immunosensor. Later, a novel electrochemicalImmunosensors are important analytical tools based on immunosensor for human chorionic gonadotrophin (hCG)the detection of the binding event between antibody and was developed by the same group via the immobilization
antigen. The recent development of immunoassaytechniques focused in most cases on decreasing analysistimes, improving assay sensitivity, simplification andautomation of the assay procedures, low-volume analysis.Among types of immunosensors, electrochemicalimmunosensors are attractive tools and have received
achieve excellent detection limits with small analytevolumes. Furthermore, the availability of a variety of newmaterials with unique properties at nanoscale dimension,such as AuNPs, has attracted widespread attention intheir utilization for the bioassay, especially forelectrochemical detection. Recently, several novelstrategies have been proposed to develop electrochemicalimmunosensors with high sensitivity using AuNPs. A
stripping voltammetry technology. A low detection limit
1
1
horseradish peroxidase-labeled electrochemical
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Fig. 5: (a) Schematic representation of preparation of an Development of environment friendly greenimmunosensing layer (b) Schematic view of methodologies have been fabricated to produceelectrochemical detection of mouse IgG or biologically benign gold nanoparticles labeledPSA137 withbiologically relevant molecules. Furthermore, the gold
of hCG on AuNPs doped three-dimensional (3D) sol–gel in vivo biodistribution. Additionally, gold nanoparticlesmatrix. The 3D organized composite structure was were conjugated with cancer seeking peptides to impartprepared by assembling AuNPs into a hydrolyzed target specificity in hybrid gold nanoparticles for their(3-mercaptopropyl)-trimethoxysilane sol–gel matrix, which potential applications in cancer imaging and therapy. Goldshowed good biocompatibility. After the interfacial nanoparticles trapped in an agarose matrix were evaluatedcompetitive immunoreaction, the formed HRPlabeled for their SERS properties with DNA nucleosides forimmunoconjugate showed good enzymatic activity for the possible biosensor applicationsoxidation of ophenylenediamine by H2O2. Theimmunosensor showed good precision, high sensitivity, REFERENCEacceptable stability and reproducibility. Label-freeelectrochemical immunosensors using AuNPs as 1. Park, J.H., Y.T. Lim, O.O. Park, J.K. Kim, J.W. Yu andenhancing sensing component have been the focus of Y.C. Kim, 2004. Polymer/Gold nanoparticleintense research due to their simplicity, speedy analysis nanocomposite light-emitting diodes: Enhancementand high sensitivity. The technique is mainly based on of electroluminescence stability and quantumthe detectionof a change in physical properties as a result efficiency of blue-light-emitting polymers. Chemistryof antibody–antigen complex formation. The direct of Materials, 16(4): 688.determination of immunospecies by detecting the change 2. Narayanan, R. and M.A. El-Sayed, 2005. Catalysisof impedance caused by immunoreactions has been with transition metal nanoparticles in colloidaldemonstrated. A simple and sensitive labelfree solution: Nanoparticle shape dependence andelectrochemical immunoassay electrode for detection of stability. Journal of Physical Chemistry B,carcinoembryonic antigen (CEA) has been developed by 109(26): 12663.Yao’s group. CEA antibody (CEAAb) was covalently 3. Aslan, K., Z. Jian, J.R. Lakowicz and C.D. Geddes,attached on glutathione (GSH) monolayer-modified 2004. Saccharide sensing using gold and silverAuNPs and the resulting CEAAb-AuNPs nanoparticles - a review. Journal of Fluorescence,bioconjugates were immobilized on Au electrode by 14(4): 391.electrocopolymerization with o-aminophenol (OAP). 4. Riviere, C., F.P. Boudghene, F. Gazeau, J. Roger,Electrochemical impedance spectroscopy studies J.N. Pons, et al., 2005. Iron oxide nanoparticle-labeleddemonstrated that the formation CEA antibody–antigen rat smooth muscle cells: Cardiac MR imaging forcomplexes increased the electron-transfer resistance of cell graft monitoring and quantitation. Radiology,[Fe(CN) ]3-/4- redox pair at the poly-OAP/CEAAb- 235(3): 959.6
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CONCLUSION
Gold nanoparticles are currently being utilized inseveral technological applications and are gainingpopularity as a form of counter measures against manyodds beared through conventional means. As a naturalmaterial, gold is known to be safe to man and producelittle to no allergic reactions when tested for curingvarious diseases. Its wide and beneficial applications arebecoming more and more demanding. This is sure a verypromising element to make our living long lasted gold.
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