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ArticleJournal of

Biomedical NanotechnologyVol 12 357ndash365 2016

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Gold Nanoparticles-Based Barcode Analysis forDetection of Norepinephrine

Jeung Hee An1 Kwon-Jai Lee2 and Jeong-Woo Choi3lowast1Division of Food Bioscience Konkuk University Chungju 380-701 Korea2Department of Advanced Materials Engineering Daejeon University Daejeon 300-716 Korea3Department of Chemical and Biomolecular Engineering Sogang University Seoul 121-742 Korea

Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmittersWe evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopamin-ergic cells Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neuro-transmitters with surface-enhanced Raman spectroscopy (SERS) and provides polymerase chain reaction (PCR)-likesensitivity This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both withDNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodiesThe aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugatedbarcode DNA The DNA barcodes are then identified by SERS and PCR analysis The concentration of norepinephrinein dopaminergic cells can be readily detected using the bio-barcode assay which is a rapid high-throughput screeningtool for detecting neurotransmitters

KEYWORDS Bio-Barcode Assay Gold Nanoparticles Norepinephrine Surface-Enhanced Raman Spectroscopy

INTRODUCTIONCatecholamines which influence a variety of motivatedbehaviors attention span and neuronal plasticity play acritical role in learning and memory1 The most abun-dant catecholamines in the human body are dopamineadrenaline and norepinephrine (NE)2 In particular NE isa catecholamine neurotransmitter that acts as a biochemi-cal messenger in mammalian central nervous systems andin neuronal cells Increased NE causes neuronal deathin Alzheimerrsquos disease3 and the loss of NE in neuronalcells in the brain is associated with Parkinsonrsquos disease4

Increased plasma NE is associated with coronary heartdisease5 and with muscle sympathetic nerve traffic inhuman obesity6 In addition NE is involved in the occur-rence and development of cardiac hypertrophy throughthe activation of myocardial adrenergic receptors on cellmembranes in myocardial tissue7 Furthermore an over-commitment to work is associated with lower NE secretionand is known to produce psychosocial stress in humans8

lowastAuthor to whom correspondence should be addressedEmail jwchoisogangackrReceived 14 November 2014Accepted 4 July 2015

whereas increased 24 h urinary NE excretion is a symp-tom of depression and anxiety in middle-aged women9

Thus accurate determination of NE levels is crucial froma clinical point of viewSeveral methods have been employed for the selec-

tive determination of NE including microdialysis10 end-column chemiluminescence11 high-performance liquidchromatography capillary electrophoresis coupled withamperometric detection12 ion chromatography with directconductivity detection13 micellar electrokinetic capil-lary chromatography with UV absorbance detection14

and voltammetry15 A novel ultrahigh-sensitivity tech-nique known as the bio-barcode amplification assay wasrecently reported16 however no studies have reportedthe detection of NE from dopaminergic cells using thismethodThe bio-barcode DNA assay enables highly sensitive

detection of proteins and nucleic acids16 This methodinvolves two types of particles magnetizable spheres mod-ified with a functional group that has an affinity for thetarget of interest and nanoparticles modified with a secondgroup that has an affinity for the same target along with anoligonucleotide barcode DNA that can act as a reporter forthe target of interest1718 The limit of detection of proteins

J Biomed Nanotechnol 2016 Vol 12 No 2 1550-7033201612357009 doi101166jbn20162185 357



Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine An et al

using this technique is approximately 30 attomolar (aM)17

A key aspect of the bio-barcode DNA assay is the use ofgold nanoparticles which enables simultaneous loading ofdetection antibodiesprobes and a large quantity of barcodeDNA per gold nanoparticle19 Because gold nanoparticlesare readily coupled with both antibodies and DNA thistechnique avoids the complicated preparation of antibody-DNA conjugates required in immuno-polymerase chainreaction (immuno-PCR)19 In addition the large ratio ofDNA to antibody (typically 100ndash3001) on the functional-ized gold nanoparticles further improves the sensitivity ofthe bio-barcode DNA assay19

In addition surface-enhanced Raman spectroscopy(SERS) using metal nanoprobes has shown promise as adetection method by increasing the sensitivity of Ramanspectroscopy20 SERS enhances the Raman signal intensityby a factor of 106 to 1014 thus allowing detection of pico-to femtomolar amounts of biomolecules21 Functional-ized SERS nanoparticles in the presence of Raman-activemolecules are typically used for the detection sensingor imaging of biological samples such as DNA proteinscells and tissues22 In particular SERS has been used toconfirm the viability of substrates such as microlithograph-ically prepared silver posts electron-beam lithographicallyproduced arrays of elongated gold nanoparticles litho-graphically produced triangular nanoparticle arrays gold-nanostructured films deposited on polystyrene colloidalcrystal templates and gold template particles grafted ontosilanized glass2324 Additionally the chemical synthesis ofsilver or gold SERS substrates has progressed rapidly inrecent years owing to the special optical properties of thesubstrates and their potential application to plasmonics andsensors7

In this study we present the first example of NEdetection with a bio-barcode assay in the context of anestablished Parkinsonrsquos disease marker We show that themodified bio-barcode method can detect NE and this issignificant because NE is recommended as the most effec-tive marker for diagnosing and monitoring patients withbrain disease

EXPERIMENTAL DETAILSDNA Oligonucleotides Bio-Barcode DNA andPCR PrimersThe sequences of the thio-capped oligonucleotide and thebio-barcode DNA were 5prime-CCA CAC TGC CGG ATGTGG ATT TAA CCT TTC TGC TAA TGT GTT-(A)10-(CH23ndashSHndash3

prime and 5primendashSHndashAAC ACA TTA GCA GAAAGG TTA AAT CCA CAT CCG GCA GTG TGG-3respectively The forward (F5-AAC ACA TTA GCA GAAAGG TTA AAT CCA CAT-3) and reverse (R5-ATG TGGATT TAA CCT TTC TGC TAA TGT GTT-3prime) primersfor amplifying the bio-barcode DNA were synthesized byBioneer (Seoul Korea)

Preparation of Magnetisable SpheresTosyl-activated magnetic Dynabead (MyOnetrade

Dynabeadsreg M-280 Invitrogen Inc Carlsbad CA USA)were heavily functionalized with a mixture of rabbitpolyclonal antibodies (Abcam Cambridge UK) TheDynabeadsreg M-280 50 are 10 m diameter magneti-zable superparamagnetic polystyrene beads consistingof nanometer-sized iron oxide particles embedded in apolymer matrix Briefly 10 L of NE polyclonal antibody(1 mgmL) 20 L magnetizable spheres 84 L of 3 M(NH42SO4 and 66 L borate buffer were combined in a02 mL PCR tube and incubated at 37 C at 1400 oscil-lationsmin for 24 h The magnetizable spheres wereseparated magnetically after incubation To block anyremaining active sites on the spheres 250 L blockingbuffer consisting of phosphate-buffered saline (PBS pH74) with 05 bovine serum albumin (BSA) and 005Tween 20 was added to the magnetic spheres and themixture incubated at 37 C for 24 h Following incuba-tion the magnetizable spheres were once again separatedmagnetically and washed twice with 1 mL magnetic-probesolution containing PBS (pH 74) 01 BSA and 005Tween 20

Preparation of Functionalized Gold ProbesThe antibody- and thiol-capped oligonucleotides wereconjugated onto gold nanoparticles using a previ-ously described one-step method25 The antibody- andthiol-capped-oligonucleotide-conjugated gold nanoparti-cles were re-suspended in 4 mL of PBS Bio-barcodeDNA (08 OD) was added and hybridized for 4 h at roomtemperature The solution was centrifuged (13000times g30 min) at 4 C and the resultant NE-nanoparticle-dsDNAcomplexes were re-suspended in a solution of 015 M NaCland 001 MPBS and stored at 4 C The antibody- andDNA-conjugated gold nanoparticles were detected using aUV-visible spectrophotometer

Capture of NE in SH-SY5Y Cells by theBio-Barcode DNA AssaySH-SY5Y cells were cultured in the presence of CO2 (5)at 37 C in Dulbeccorsquos modified eaglersquos medium supple-mented with fetal bovine serum (10) and 05 antibi-otics Supernatant was collected from cell cultures andused directly for bio-barcode DNA analysisIn a typical experiment an immunoassay was conducted

consisting of generation of a magnetic immunoprobenorepinephrinegold immunoprobe sandwich followed bycollection of the DNA bio-barcodes by magnetic separa-tion and thermal dehybridization25 Protein extraction wasperformed by protease K enzyme analysis After incuba-tion for 1 h at 50 C the supernatant of the reactionmixture was used as a template for PCR or conjugatedto complementary DNA in nanogold plates (see belowSection Preparation of Complementary DNA in Nanogold

358 J Biomed Nanotechnol 12 357ndash365 2016



An et al Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine

Plate for SERS Detection) NE was detected by RT-PCR for the matrix gene according to a procedure pub-lished elsewhere with some modifications8 The obtainedRT-PCR products were analyzed using 2 agarose gelelectrophoresis DNA was also detected by SERS Com-plementary DNA was detected by its Raman signal how-ever non-complementary DNA did not exhibit a Ramansignal

Preparation of Complementary DNA in NanogoldPlate for SERS DetectionWell-ordered nanoporous alumina masks were preparedfrom aluminum foil (9999 100 m in thickness) viaa two-step anodization process26 An indium tin oxide(ITO) glass substrate was placed on the alumina maskand the two were turned upside down The filter paperwas then carefully peeled from the alumina mask Thealumina mask with through-holes and the ITO glass wereplaced on a sample holder in an evaporator systemGold was deposited on the ITO substrate through thepores of the nanoporous alumina mask using a thermalevaporator (ULVACVPC-260) with a vacuum pressure of3times 10minus6 Torr and an evaporation rate of approximately01 Aring sminus1 After gold deposition the alumina mask placedon the ITO substrate was dissolved for 5 min in 10 wtvNaOH and then the substrate was rinsed three times in dis-tilled water After the pre-treatment a well-ordered com-plementary DNA (5primendashSHndashAAC ACA TTA GCA GAAAGG TTA AAT CCA CAT CCG GCA GTG TGG-3) wasfabricated directly on the freshly cleaned gold electrode

Raman SpectroscopyThe biocomposition of living cells and the effect of neuro-toxic agents on SH-SY5Y cells were investigated by SERSusing Raman NTEGRA spectra (NT-MDT ZelenogradMoscow Russia) The maximum scan range (XYZ) was100 mtimes100 mtimes6 m the resolution of the spectrom-eter was 200 nm in the XY plane and 500 nm along theZ axis Raman spectra were recorded using an NIR laseremitting light at a wavelength of 785 nm Ten scans of 1 seach from 500 to 1600 cmminus1 were recorded and the meanof those scans was used in the analysis

RESULTS AND DISCUSSIONSynthesis and Characterization ofAntindashNE-Conjugated Barcode DNAThe bio-barcode assay utilized 60 nm Au nanoparticleprobes and tosyl-activated magnetic Dynabeads each co-functionalized with a polyclonal antibody that recognizedthe target antigen (Fig 1) The Au nanoparticle probesare modified for detection of antibody and barcode DNAstrands (43 bpparticle) Note that half of the barcode foreach target is the target-reporting oligonucleotide probewhile the other half is identical for all the barcodes and

is in effect a universal sequence The Au nanoparticleprobes were stored at a concentration of 08 OD with theexcess barcode DNA removed prior to use by washing in015 M NaCl 0025 Tween 20 01 BSA and 10 mMPBS (pH 74) During the assay the target antigens arecaptured in solution by magnetizable spheres each conju-gated with a monoclonal antibody specific for an epitopeof the target antigen different from the one recognizedby the gold probe17 The target-magnetizable sphere com-plexes are sandwiched between the antibody-conjugatedAu nanoparticle probes (Fig 1) These complexes are iso-lated using a magnetic field washed and the barcode DNAstrands are released from the nanoparticle probes by a lig-and exchange process induced by the addition of dithio-threitol The bio-barcode strands are then identified byPCR and SERSWe constructed nanogold substrates containing approxi-

mately equal numbers of Au nanostructures that containedSERS-active spots Au dots with a controlled size anduniform spatial distribution for use as a nanobioplatformfor the SERS detection plate were achieved reproduciblyby using nanoporous alumina masks The close-packedhexagonal pore array of the alumina plays a very importantrole in determining the ordering of the nanostructures26 Toachieve this a long-range-ordered ultrathin alumina maskwith through-holes was placed on an ITO glass substrateThe mask could bind to the substrate via van der Waalsinteractions27 The average diameter of the openings in thenanoporous alumina was 73plusmn5 nm as measured by scan-ning electron microscopy (SEM) analysis (Fig 2) A rep-resentative SEM image of the Au nanosubstrate arraysformed on the ITO glass after removal of the aluminamask is shown in Figure 2 The uniform nanostructuresform periodic patterns with an average distance of sepa-ration of 100 nm It could be clearly seen that the uni-form nanoporous structures were formed as a replica of themask in spite of the problems inherent in their growth onthe ITO glass The average diameter of the gold nanoislandarrays was approximately 70plusmn5 nm28 An SEM image ofthe immobilized colloidal gold nanoparticles showed thatthe average particle diameter was about 73 nm (Fig 2)Most gold nanoparticles self-assembled near each other byforming randomly distributed one-dimensional patterns29

Detection of NE Using Barcode ProbesMany factors can limit the performance of DNA-barcode-based immunoassays These factors include the type ofparticles or antibodies the attachment chemistry and theeffectiveness of the removal and washing steps Of thesethe immunological affinity of the immunoprobes towardtheir analytical target plays the most important role Inour study the concentration range required for detectioncould easily be differentiated from that of a negative con-trol (0 ngmL) To determine this the magnetizable sphereimmunoprobes were successively incubated with increas-ing concentrations of NE After the incubation period the

J Biomed Nanotechnol 12 357ndash365 2016 359




Figure 1 Schematic diagram of the surface-immobilized bio-barcode assay protocol

samples were treated with protease K and the barcodeDNA was eluted Finally 50 L DNA-free water wasadded to dissolve the DNA for PCR analysis (Fig 3(a))A linear relationship was observed between the target con-centration and NE barcode DNA PCR signal intensity overfive different NE concentrations ranging from 1 to 100 nM(Fig 3(a)) It should be noted that inadequate washingmight produce false negative results in any immunoassayHowever both washing solutions were demonstrated tobe free of NE after washing was repeated three times asshown by the absence of any PCR signal at the 0 ngmLconcentration (Fig 3(a))The NE-conjugated DNA formed through a self-

assembled gold substrate was examined by Raman spec-troscopy SERS phenomena involving Au nanoparticles

Figure 2 Scanning electron microscope (SEM) images of the alumina mask gold nano-parttern fabricated on an indium tinoxide (ITO) glass substrate and anti-norepinephrine-conjugated gold nanoparticles

have been well-documented because their signals can beidentified at the single-molecule level by association withthe resonance effect of the adsorbed molecule30 UsingSERS we detected signal levels corresponding to four dif-ferent NE concentrations ranging from 0 to 100 nM andthe intensity of the NE barcode DNA signals increasedin a dose-dependent manner (Fig 3(b)) We obtainedSERS spectra for a range of wave numbers from 500 to1600 cmminus1 (Fig 3(b)) The peaks that appeared were thosemost affected by adsorption along with the conventionalRaman spectrum of the test molecule excited by a 785 nmlaser All conventional Raman spectra were recorded usingexcitation lines Furthermore the SERS spectra recordedfrom different spots on the surface were identical whichdemonstrated the reproducibility of the SERS substrate





Figure 3 Electropherogram illustrating validation of the immunoprobe by PCR to detect norepinephrine Lane 1 negative con-trol lane 2 1 nM lane 3 10 nM lane 4 50 nM lane 5 100 nM and lane 6 DNA ladder All PCR amplifications were for 35 cycles andthe resulting PCR products (20L) were electrophoresed on a 2 agarose gel and visualized with ethidium bromide (b) Ramanspectra of the DNA barcodes for different norepinephrine concentrations identified by surface-enhanced Raman spectroscopy(c) linear graph of Raman bands at 729 1375 1485 and 1576 cmminus1 at various norepinephrine concentrations and (d) linear plotof Raman bands at 1375 cmminus1 at various norepinephrine concentrations R2 = 09801 Y = 11913+12635X

generated for this analysis The Raman spectrum of thegold substrate alone was observed at 1383 cmminus1 whichwas similar to the pattern of the surface with singlestranded non-complementary DNA The Raman spectrumof the NE barcode DNA base-paired to the complemen-tary DNA on the substrate showed broad bands at 670729 847 1078 1092 1179 1375 1485 and 1578 cmminus1

(Fig 3(b)) Raman peaks for DNA components were foundat 670 (guanine) 729 (adenine) 847 (deoxyribose phos-phodiester) 1078 (phophodioxy) 1179 (thymine cyto-sine) 1375 (thymine) 1485 (adenine thymine cytosine)and 1578 cmminus1 (adenine N6H2 df)31ndash35 A detection limitof 1 nM NE could be estimated using a signalnoise (SN)ratio of 3 The band at 1375 cmminus1 was the most intenseand as such was subsequently used to record the SERSspectra it also deviated from the barcode sequence ofthe gold particles34 When the concentration of antigen-NE was greater than 100 nM the intensity of the SERSsignal at 1375 cmminus1 increased rapidly (Fig 3(c)) Therewas a good linear relationship between the Raman inten-sity of the 1375 cmminus1 peak and the NE concentrationfrom 1times 10minus9 to 1times 10minus7 M with a correlation coef-ficient of 09801 (Fig 3(c)) The regression equation

was Y = 11913+ 12635X (Y Raman intensity X NEconcentration)Finally we developed a calibration model that pre-

dicted the antigen concentration between 1 and 100 nM(Fig 3(d)) Decreasing the concentration of NE causeda decrease in the relative intensities of the Ramanpeaks at 729 (R2 = 09520) 1179 (R2 = 09764)1485 (R2 = 09835) and 1576 (R2 = 09835) cmminus1 whichcorresponded to the DNA bases In addition the regressionequations of 729 1179 1485 and 1576 cmminus1 were Y =10465+11185X Y = 10085+10955X Y = 9644667+9870005X and Y = 85513333+ 8765X respectivelyThus our result showed that the SERS response increasedlinearly with increasing concentrations of NE The resultssuggested that an NE-conjugated bio-barcode DNA orderedon Au nano-patterns is an effective tool for neurotransmit-ter detection by SERS-based measurements Importantlysimilar patterns were found between the SERS spectra andthe PCR image in terms of the dose-dependent detection ofNE Therefore our results demonstrated that SERS mightbe an important tool for monitoring neurotransmitters suchas dopamine epinephrine and NE





Detection of NE in SH-SY5Y Cells byBarcode AnalysisTo show that this assay has physiological relevance it wasnecessary to verify that the immunoprobes captured theNE secreted by dopaminergic cells such as SH-SY5Y cells(Fig 4(a)) SERS analysis was exploited for this pur-pose as a straightforward approach to validate the useful-ness of the immunoprobe Failure to capture NE could becaused by a blocking effect of the microbeads or by non-specific interactions between the beads and NE Potentialnon-specific interactions of the immunoprobe with othercatecholamines are also an important confounding fac-tor and should be identified to evaluate the specificityof the immunoprobes To evaluate the clinical applicationpotential of the proposed method recovery experimentswere performed NE extracts were obtained from 1times 106

cellsmL samples prepared from SH-SY5Y cells and theNE recovery ranged from 967 to 103 (Fig 3(d))We obtained SERS spectra for the SH-SY5Y cells

for a range of wavenumbers from 500 to 1600 cmminus1The peaks that appeared in these spectra were mostlythose affected by adsorption along with the conventionalRaman spectrum of the test molecule excited by a 785nm laser (Fig 4(a)) The Raman spectrum of SHndashSY5Ycells showed Raman bands at 670 729 847 1078 10921179 1375 1485 and 1578 cmminus1 Their bands were sim-ilar to those seen in the SERS spectra of the DNA base(Fig 4(a)) peak identifications were as described above

Figure 4 (a) Surface-enhanced Raman spectroscopy (SERS) spectra of SH-SY5Y cells exposed to 500 nM polychlorinatedbiphenyls (PCBs) rotenone 6-hydroxydopamine (6-ODHA) or bisphenol for 24 h (b) Changes in the DNA content of Ramanpeaks from SH-SY5Y cells treated with 500 nM PCB 6-ODHA rotenone or bisphenol (c) Cell toxicity analysis of control cellsand SHndashSY5Y cells treated with PCB 6-ODHA rotenone or bisphenol

The Raman spectra of NE showed similar patterns in SH-SY5Y cells The cellular concentration of NE was calcu-lated at Raman peaks of 1375 cmminus1 and it is shown inFigure 4(b) Thus the concentration of NE secreted byuntreated SH-SY5Y cells was determined to be 112times10minus8 M using our bio-barcode analysis (Fig 3(d))Four neurotoxins polychlorinated biphenyls (PCBs)

rotenone 6-hydroxydopamine (6-ODHA) and bisphenolwere selected as representative oxidative-stress-inducingdrugs to study their effects on NE in SH-SY5Y dopamin-ergic cells using SERS-based DNA bio-barcode analysisThe measured Raman spectra indicated that many bio-chemical changes occurred after treatment with the neu-rotoxins and differences in biochemical composition wereevident particularly at the Raman peaks associated withDNA Overall treatment with the neurotoxins decreasedthe relative intensities of the Raman peaks In particularalthough the 1375 cmminus1 bands could be readily observedthe other peaks such as at 670 1485 and 1579 cmminus1 werenot detected in the PCB rotenone or 5-OHDA treatedcells Also the cellular concentration of NE was calculatedat Raman peaks of 1375 cmminus1 (Y = 11913+ 12635X)The concentration of NE was found to be 562times10minus9 Mafter PCB treatment 556times 10minus9 M in rotenone-treatedcells 427times10minus9 M in 5-OHDA ndashtreated cells and 312times10minus9 M in bisphenol A-treated cells (Fig 4(b))Generally PCB exposure is known to pose numerous

health risks indeed several studies have shown PCBs to





be detrimental to the central nervous system resulting inmotor and cognitive deficits and Parkinsonrsquos disease3536

A common finding has been a significant reduction in braindopamine and tyrosine hydroxylase and dopamine trans-porters levels following exposure to PCBs3637 Rotenoneis a common pesticide and is a well-characterized specificinhibitor of complex I of the mitochondrial respiratorychain Rats chronically exposed to rotenone developed theneuropathological and behavioral symptoms of Parkinsonrsquosdisease resulting from the induction of apoptosis andacceleration of -synuclein formation in pharmacody-namic models in vivo and in vitro39 Furthermore 6-OHDAis an oxidative neurotoxin that injures dopaminergic neu-rons in vivo and in vitro It induces catecholaminergiccell death by three primary mechanisms production ofreactive oxygen species formation of hydrogen perox-ide and direct inhibition of the mitochondrial respiratorychain39 Exposure to bisphenol A (BPA) at environmen-tally relevant levels (either 25 or 250 ng per kg of bodymass) caused altered brain sexual differentiation in ratoffspring40 Specifically decreases in the numbers of tyro-sine hydroxylase (TH) neurons were observed in the ros-tral periventricular preoptic area of BPA-exposed femaleoffspring and this removed sexual differences in the THneurons41

To verify the hypothesis that a decrease in the Ramanpeak current is associated decreased cell viability tetra-zolium dye (MTT) analysis was performed (Fig 4(c))The viability of cells decreased after treatment withPCB rotenone 6-ODHA and bisphenol at 500 nM con-centrations by 267 30 215 and 405 respectivelyThese results demonstrated that neurotoxins such as PCBsrotenone 6-ODHA and bisphenol accelerate apoptosisand decrease cell viability Furthermore these results veri-fied that the Raman response was in fact a good measureof the effects of the toxins on cell viability ThereforeRaman spectra are a simple way to investigate the effectsof toxins on dopaminergic cells by monitoring changes incellular physiology and viability Thus our technique canbe used to fabricate a highly sensitive low-cost neuro-transmitter sensor for detecting environmental toxins

Cell Image and Apoptosis SignalCellular toxicity following PCB treatment was determinedto be 733 in neuronal cells Therefore we also eval-uated the relevance of apoptosis in neuronal cell deathinduced by PCB using annexin V-FITC staining This anal-ysis demonstrated that PCB treatment increased the num-ber of apoptotic SHndashSY5Y cells As shown in Figure 5(a)treatment with 500 nM PCB resulted in 5966 late apop-totic cells as compared with only 018 in untreated cells

To study Bax-induced cytochrome c release duringapoptosis using a neuronal cell model we exploited thefact that Bax and cytochrome c can fluoresce after react-ing with PCBs The expression levels of cytosolic Bax andcytochrome c (Fig 5(b)) were elevated in the PCB-treated

Figure 5 (a) Apoptosis profile of annexin V-FITCPI stainingfor detection of apoptotic cells in SH-SY5Y cells After treat-ment with PCB (0 or 500 nM) cells were stained with annexinV-FITCPI and subjected to flow cytometry (b) Immunoflu-orescence image of Bax and cytochrome c in PCB-treatedSH-SY5Y cells Cells were fixed in 4 paraformaldehyde(15 min) rinsed in PBS and probed with antibodies specificfor Bax and cytochrome c using standard immunocytochem-istry techniques Images were acquired using a Zeiss LSM510Axioplan-2 upright confocal microscope using the LSM imag-ing software (Carl Zeiss Jena Germany) Scale bar 20 mCon control PCB polychlorinated biphenyls FITC fluores-cein isothiocyanate PI phosphatidylinositol

SHndashSY5Y cells as compared to untreated control cells(Fig 5(b)) Furthermore Bax and cytochrome c expressionin PCB-treated cells exhibited greater intensity comparedto the control cells and the cytosol of these was stronglycondensed Together these results demonstrated the pres-ence of PCB-stimulated apoptosis in SH-SY5Y cells andthat expression of Bax and cytochrome c was increased inPCB-treated dopaminergic cells This suggests that oxidiz-ing reactions of Bax and cytochrome c were involved inPCB-induced SH-SY5Y cell toxicity

CONCLUSIONSGold nanoparticles modified with an antibody and dsDNAoligonucleotides were used for an immunosorbent bio-barcode assay to detect trace amounts of NE The assay





is based on a sandwich immunoassay and DNA bar-code detection using SERS Magnetizable spheres withantibodies and gold nanoparticles that are encoded withDNA can sandwich the target protein captured by thenanoparticle-bound antibodies The aggregate sandwichstructures are magnetically separated from solution andtreated to remove the conjugated barcode DNA allowingthe DNA barcodes to be identified by SERS and PCRanalysis Increasing the concentration of NE resulted in aparallel increase in the relative intensities of the Ramanpeaks at 670 729 847 1078 1092 1179 1375 1485and 1578 cmminus1 which correspond to the DNA bases Theimmunosorbent bio-barcode assay using modified goldnanoparticles was demonstrated to be sensitive enough todetect NE at a range of 1 to 100 nM Treatment with neu-rotoxins decreased the relative intensities of the Ramanpeaks at 1375 cmminus1 which were used to calculate the NEconcentrations secreted by SH-SY5Y cells The concen-tration of NE was found to be 562times 10minus9 M after PCBtreatment 556times10minus9 M in rotenone-treated cells 427times10minus9 M in 5-OHDA ndashtreated cells and 312times10minus9 M inbisphenol A-treated cells Therefore the NE concentrationin dopaminergic cells can be detected easily and rapidlyusing this bio-barcode assay which can be considered arapid high-throughput screening tool for detecting neuro-transmitters such as NE and dopamine

Acknowledgments This research was supported byBasic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistry of Education (2014R1A1A2058817) and bythe National Research Foundation of Korea (NRF)grant funded by the Korea government (MSIP) (No2014R1A2A1A10051725)

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23 M Baia F Toderas L Baia J Popp and S Astilean Prob-ing the enhancement mechanisms of SERS with p-aminothiophenolmolecules adsorbed on self-assembled gold colloidal nanoparticlesChem Phys Lett 422 127 (2007)





24 K W Fujiwara H Itoh E H Nakahama and N Ogawa Measure-ment of antibody binding to protein immobilized on gold nanopar-ticles by localized surface plasmon spectroscopy Anal BioanalChem 386 639 (2006)

25 J H An W A El-Said and J W Choi Detection of dopamine indopaminergic cell using nanoparticles-based barcode DNA analysisJ Nananosci Nanotech 12 764 (2012)

26 M Jung W A El-Said and J W Choi Fabrication of gold nanodotarrays on a transparent substrate as a nanobioplatform for label-freevisualization of living cells Nanotechnology 22 235304 (2011)

27 X Mei M Blumin D Kim Z H Wu Q X Guo and H ERuda Molecular beam epitaxial growth studies of ordered GaAsnanodot arrays using anodic alumina masks J Crystal Growth 251253 (2003)

28 M Jung B Y Oh and J W Choi Fabrication of Au nanodotswith 60 nm diameter on ITO glass Towards nanobiochip usingnanoporous alumina mask Ultramicroscopy 109 1006 (2009)

29 M A Kafi T H Kim C H Yea H Kim and J W Choi Effectsof nanopatterned RGD peptide layer on electrochemical detection ofneural cell chip Biosens Bioelctron 26 1359 (2010)

30 W A El-Said T H Kim H Kim and J W Choi Detection ofeffect of chemotherapeutic agents to cancer cells on gold nanoflowerpatterned substrate using surface-enhanced Raman scattering andcyclic voltammetry Biosens Bioelectron 26 1486 (2010)

31 A Barhoumi D Zhang F Tam and N J Halas Surface-enhancedRaman spectroscopy of DNA J Am Chem Soc 130 5523 (2008)

32 K Kneipp H Kneipp I Itzkan R R Dasari and M S FeldSurface-enhanced Raman scattering and biophysics J Phys Con-dens Matter 14 597 (2002)

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using this technique is approximately 30 attomolar (aM)17

A key aspect of the bio-barcode DNA assay is the use ofgold nanoparticles which enables simultaneous loading ofdetection antibodiesprobes and a large quantity of barcodeDNA per gold nanoparticle19 Because gold nanoparticlesare readily coupled with both antibodies and DNA thistechnique avoids the complicated preparation of antibody-DNA conjugates required in immuno-polymerase chainreaction (immuno-PCR)19 In addition the large ratio ofDNA to antibody (typically 100ndash3001) on the functional-ized gold nanoparticles further improves the sensitivity ofthe bio-barcode DNA assay19

In addition surface-enhanced Raman spectroscopy(SERS) using metal nanoprobes has shown promise as adetection method by increasing the sensitivity of Ramanspectroscopy20 SERS enhances the Raman signal intensityby a factor of 106 to 1014 thus allowing detection of pico-to femtomolar amounts of biomolecules21 Functional-ized SERS nanoparticles in the presence of Raman-activemolecules are typically used for the detection sensingor imaging of biological samples such as DNA proteinscells and tissues22 In particular SERS has been used toconfirm the viability of substrates such as microlithograph-ically prepared silver posts electron-beam lithographicallyproduced arrays of elongated gold nanoparticles litho-graphically produced triangular nanoparticle arrays gold-nanostructured films deposited on polystyrene colloidalcrystal templates and gold template particles grafted ontosilanized glass2324 Additionally the chemical synthesis ofsilver or gold SERS substrates has progressed rapidly inrecent years owing to the special optical properties of thesubstrates and their potential application to plasmonics andsensors7

In this study we present the first example of NEdetection with a bio-barcode assay in the context of anestablished Parkinsonrsquos disease marker We show that themodified bio-barcode method can detect NE and this issignificant because NE is recommended as the most effec-tive marker for diagnosing and monitoring patients withbrain disease

EXPERIMENTAL DETAILSDNA Oligonucleotides Bio-Barcode DNA andPCR PrimersThe sequences of the thio-capped oligonucleotide and thebio-barcode DNA were 5prime-CCA CAC TGC CGG ATGTGG ATT TAA CCT TTC TGC TAA TGT GTT-(A)10-(CH23ndashSHndash3

prime and 5primendashSHndashAAC ACA TTA GCA GAAAGG TTA AAT CCA CAT CCG GCA GTG TGG-3respectively The forward (F5-AAC ACA TTA GCA GAAAGG TTA AAT CCA CAT-3) and reverse (R5-ATG TGGATT TAA CCT TTC TGC TAA TGT GTT-3prime) primersfor amplifying the bio-barcode DNA were synthesized byBioneer (Seoul Korea)

Preparation of Magnetisable SpheresTosyl-activated magnetic Dynabead (MyOnetrade

Dynabeadsreg M-280 Invitrogen Inc Carlsbad CA USA)were heavily functionalized with a mixture of rabbitpolyclonal antibodies (Abcam Cambridge UK) TheDynabeadsreg M-280 50 are 10 m diameter magneti-zable superparamagnetic polystyrene beads consistingof nanometer-sized iron oxide particles embedded in apolymer matrix Briefly 10 L of NE polyclonal antibody(1 mgmL) 20 L magnetizable spheres 84 L of 3 M(NH42SO4 and 66 L borate buffer were combined in a02 mL PCR tube and incubated at 37 C at 1400 oscil-lationsmin for 24 h The magnetizable spheres wereseparated magnetically after incubation To block anyremaining active sites on the spheres 250 L blockingbuffer consisting of phosphate-buffered saline (PBS pH74) with 05 bovine serum albumin (BSA) and 005Tween 20 was added to the magnetic spheres and themixture incubated at 37 C for 24 h Following incuba-tion the magnetizable spheres were once again separatedmagnetically and washed twice with 1 mL magnetic-probesolution containing PBS (pH 74) 01 BSA and 005Tween 20

Preparation of Functionalized Gold ProbesThe antibody- and thiol-capped oligonucleotides wereconjugated onto gold nanoparticles using a previ-ously described one-step method25 The antibody- andthiol-capped-oligonucleotide-conjugated gold nanoparti-cles were re-suspended in 4 mL of PBS Bio-barcodeDNA (08 OD) was added and hybridized for 4 h at roomtemperature The solution was centrifuged (13000times g30 min) at 4 C and the resultant NE-nanoparticle-dsDNAcomplexes were re-suspended in a solution of 015 M NaCland 001 MPBS and stored at 4 C The antibody- andDNA-conjugated gold nanoparticles were detected using aUV-visible spectrophotometer

Capture of NE in SH-SY5Y Cells by theBio-Barcode DNA AssaySH-SY5Y cells were cultured in the presence of CO2 (5)at 37 C in Dulbeccorsquos modified eaglersquos medium supple-mented with fetal bovine serum (10) and 05 antibi-otics Supernatant was collected from cell cultures andused directly for bio-barcode DNA analysisIn a typical experiment an immunoassay was conducted

consisting of generation of a magnetic immunoprobenorepinephrinegold immunoprobe sandwich followed bycollection of the DNA bio-barcodes by magnetic separa-tion and thermal dehybridization25 Protein extraction wasperformed by protease K enzyme analysis After incuba-tion for 1 h at 50 C the supernatant of the reactionmixture was used as a template for PCR or conjugatedto complementary DNA in nanogold plates (see belowSection Preparation of Complementary DNA in Nanogold











































































































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