Exploiting the Anti-Aggregation of Gold Nanostars ... - ACS Publications

Subscriber access provided by AUBURN UNIV AUBURN

Article

Exploiting the Anti-Aggregation of Gold Nanostars for Rapid Detection of Hand, Foot and Mouth Disease Causing Enterovirus 71 using Surface-Enhanced Raman Spectroscopy Miguel Reyes, Marek Piotrowski, Swee Kim Ang, Jingqi Chan, Shuai He, Justin Jang Hann Chu, and James Chen Yong Kah Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00066 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Exploiting the Anti-Aggregation of Gold Nanostars for Rapid Detection of Hand, Foot and Mouth Disease Causing Enterovirus 71 using Surface-Enhanced Raman Spectroscopy Miguel Reyes1, Marek Piotrowski2,3, Swee Kim Ang4, Jingqi Chan5, Shuai He6, Justin Jang Hann Chu4, James Chen Yong Kah6,7* 1

Department of Materials Science and Engineering, National University of Singapore, 9 Engineering Drive 1, Blk EA, #03-09, Singapore 117575

2

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland

3

International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, 4715-330 Braga, Portugal 4

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Blk MD4, Level 5, Singapore 117597 5

6

Temasek Junior College, 22 Bedok South Rd, Singapore 469278

Department of Biomedical Engineering, National University of Singapore, 4 Engineering Drive 3, Blk E4, #04-08, Singapore 117583

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7

Page 2 of 37

NUS Graduate School for Integrative Sciences and Engineering, National University of

Singapore, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456

KEYWORDS Enterovirus 71, rapid detection, nanoparticle aggregation, surface-enhanced Raman spectroscopy (SERS), gold nanostars


2

Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT

Enterovirus 71 (EV71) is a major public health threat that requires rapid point-of-care detection. Here, we developed a surface-enhanced Raman spectroscopy (SERS)-based scheme that utilized protein-induced aggregation of colloidal gold nanostars (AuNS) to rapidly detect EV71 without the need for fabricating a solid substrate, Raman labels or complicated sample handling. We used AuNS (hydrodynamic diameter, DH of 105.12 ± 1.13 nm) conjugated to recombinant scavenger receptor class B, member 2 (SCARB2) protein with known affinity to EV71. In the absence of EV71, AuNS-SCARB2 aggregated in biological media and produced four enhanced Raman peaks at 390, 510, 670, and 910 cm-1. In the presence of EV71, the three peaks at 510, 670 and 910 cm-1 disappeared while the peak at 390 cm-1 diminished in intensity as the virus bound to AuNS-SCARB2 and prevented them from aggregation. These three peaks (510, 670 and 910 cm-1) were potential markers for specific detection of EV71 as their disappearance was not observable with a different dengue virus (DENV) as our control. Furthermore, the Raman measurements from colloidal SERS were more sensitive in probing the aggregation of AuNS-SCARB2 for detecting the presence of EV71 in protein-rich samples compared to UV-Vis spectrum measurements. With this facile “anti-aggregation” approach, we were able to detect EV71 in protein-rich biological medium within 15 min with reasonable sensitivity of 107 pfu/ml and minimal sample preparation, making this translatable for point-ofcare applications.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

INTRODUCTION The human enterovirus 71 (EV71) causing hand, foot, and mouth disease (HFMD), is a major threat across many countries

1-3

. EV71 infections mainly affect children and cause unpleasant

symptoms including ulcers in the throat, mouth, and tongue, as well as rashes on the hands and feet. Apart from these, they can also cause neurologic and systemic complications, leading to death 4. With limited vaccines only available for clinical use in China 5, rapid diagnosis is therefore paramount in containing the spread of EV71 and preventing outbreaks of HFMD. Current diagnostic techniques for EV71 infections include viral isolation 6, neutralization assays 7, and reverse transcription polymerase chain reaction (RT-PCR) 8. Whilst highly sensitive, these techniques are often slow and require a well-equipped laboratory, deeming them unsuitable for rapid point-of-care detection, especially in areas of potential outbreaks. Surface-enhanced Raman spectroscopy (SERS) is a viable technique for rapid viral detection in a non-destructive manner 9, with sensitivities down to single molecules

10-12

. Viral assays

based on SERS have been developed either by directly acquiring the Raman spectra that are characteristic of the chemical moieties on viral membrane of viruses immobilized on a metallic substrate

13-15

, or indirectly by targeting the viral surface proteins and DNA with nanoparticles

(NPs) labeled with Raman reporters, and detecting Raman signature of the reporter

16-19

, similar

to enzyme-based immunoassays. In direct SERS detection, the SERS substrates used are costly due to sophisticated fabrication despite being able to provide a rapid and simple way of detecting viruses in a sample. On the other hand, indirect SERS detection involves complicated sample handling and washing steps, making them unsuitable for point-of-care applications, which require minimal user intervention


4

Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

. Therefore, a simple, low cost, point-of-care approach for rapid detection of EV71 is presently

not available with existing detection strategies based on SERS. Such an approach would allow HFMD to be rapidly screened onsite for suspected outbreaks. Towards this end, we developed a novel SERS-based virus detection scheme based on proteininduced aggregation of colloidal gold nanostars (AuNS), a phenomenon that is largely considered a nuisance in nanomedicine applications 21-23. This scheme did not require fabrication of a solid substrate, Raman labels or complicated sample handling techniques. Here, we used colloidal gold nanostars (AuNS) conjugated to recombinant scavenger receptor class B, member 2 (SCARB2) proteins, a cell surface receptor found in mammalians cells that binds to EV71 24, whose colloidal stability was mediated by the presence of EV71. In the absence of EV71, AuNSSCARB2 formed aggregates in biological media and produced enhanced Raman peaks. However, in the presence of EV71, these peaks were diminished since the bound EV71 on AuNS-SCARB2 prevented them from aggregating (Figure 1). With this facile “anti-aggregation” approach, we were able to detect EV71 in protein-rich biological medium within 15 min with reasonable sensitivity and minimal sample preparation, making this translatable for point-of-care applications.


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

Figure 1. SERS viral detection scheme for EV71 based on anti-aggregation of gold nanostars. In the absence of EV71, AuNS-SCARB2 particles formed aggregates due to non-specific interactions with proteins in the biological medium that induced aggregation to produce enhanced Raman peaks. In the presence of EV71, the EV71 bound to AuNS-SCARB2 and prevented them from aggregating, thereby causing the Raman peaks to be diminished.

EXPERIMENTAL SECTION Materials and Reagents Gold (III) chloride (HAuCl4), silver nitrate (AgNO3), L-ascorbic acid (C6H8O6), sodium chloride (NaCl), Tris hydrochloride (TrisHCl), and Dulbecco’s modified Eagle medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO). E. coli (strain BL21(DE3)) cells were obtained from EMD Millipore (Billerica, MA) and human rhabdomyosarcoma cells (CCL136) were obtained from ATCC (USA). Champion pET SUMO Expression System was purchased from Thermo Fisher Scientific (Waltham, MA). Milli-Q water (Millipore, USA) with


6

Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a resistivity of 18.2MΩ cm was used for all experiments. All chemicals and reagents were used as received.

Synthesis and Characterization of AuNS AuNS were synthesized based on modifications to the one-pot seedless protocol published previously

25

. Here, 360 µl of 10 mM gold (III) chloride (HAuCl4) and 20 µl of 10 mM silver

nitrate (AgNO3) were mixed in 10 ml H2O under vortex for 30 s in a 15 ml centrifuge tube, before adding 60 µl of 100 mM L-ascorbic acid (C6H8O6) as the reducing agent and vortexing for another 20 s. The solution turned from a faint yellow color to greenish blue almost immediately. The synthesized AuNS colloid was centrifuged twice at 300 × g for 3 h, resuspended in 2 ml H2O, and then stored at 4° C until further use. The final AuNS concentration is ≈ 50 pM based on mass calculations assuming that the Au3+ was fully reduced. The absorption spectra of AuNS were obtained using a UV-Vis spectrophotometer (MultiSkan GO, Thermo Fisher Scientific Inc., USA). The hydrodynamic diameter (DH) of AuNS was obtained by dynamic light scattering (DLS) using a Zetasizer (Nano ZS, Malvern, UK), and their actual size and morphology were characterized using transmission electron microscopy (TEM) (JEM-1220, JEOL Ltd, Japan) and scanning electron microscopy (SEM) (Quanta 650 FEG, FEI, USA). To quantify the colloidal stability of AuNS and its conjugates, we defined an aggregation index (AI) from the ratio of absorbance at 850 nm to 700 nm, i.e. =

The surface plasmon resonance (SPR) peak of AuNS was typically ~700 nm, and its intensity correlated to the concentration of AuNS 26. Aggregation of AuNS would cause a broadening and


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

red-shift in absorbance peak, leading to an increase in the absorbance at 850 nm. Normalization to the absorbance at 700 nm allowed us to quantify AuNS aggregation in a concentrationindependent manner. A high AI value corresponds to a high degree of aggregation.

Conjugation of SCARB2 on AuNS To synthesize SCARB2 proteins, the luminal domain of SCARB2 (amino acids 35-430) were expressed with an N-terminal His-tagged SUMO (small ubiquitin-like modifier) fusion protein in E. coli (strain BL21(DE3)). The SUMO-SCARB2 proteins were then purified from the bacteria, and subsequently buffer-exchanged into Tris buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) and concentrated before treatment with SUMO protease to remove the fusion protein according to the recommended protocols from the manufacturer. The recovered SCARB2 proteins were stored at -80 °C until further use. To determine the minimum protecting amount (MPA) of SCARB2 required to form a corona around AuNS sufficient to stabilize them from salt induced aggregation, 100 µl of varying concentrations of SCARB2 (0, 10, 20-50 µg/ml) in Tris buffer (pH 7.4) with 50 mM imidazole were added to 100 µl of 50 pM AuNS, following a previously published protocol 27. The solution was then incubated for 1 h at room temperature under continuous shaking to allow passive adsorption of SCARB2 on AuNS. After incubation, 100 µl of 100 mM NaCl was added to induce AuNS aggregation. The absorption spectrum was then acquired after 10 min for all SCARB2 concentrations, and the MPA was the minimum SCARB2 concentration which resulted in no change to the AI of AuNS after addition of NaCl. In succeeding experiments, the MPA of SCARB2 (40 µg/ml) was added to AuNS and incubated at 37 °C for 1 h to form a SCARB2 corona around AuNS. Excess unbound SCARB2


8

Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were removed by repeated centrifugation at 1,900 rpm for 25 min, and the AuNS-SCARB2 was resuspended in 100 µl Dulbecco’s modified Eagle medium (DMEM). To characterize the AuNSSCARB2 prepared, its absorption spectrum, hydrodynamic diameter and Raman spectra were obtained. By comparing their peak absorption to that of synthesized AuNS, we estimated the concentration of AuNS-SCARB2 to be ≈ 20 pM.

Interaction between AuNS-SCARB2 and EV71 EV71 strain 41 (5865/SIN/000009) (GenBank accession no. AF316321) was used in this study. The EV71 samples were prepared by propagation on human rhabdomyosarcoma (RD) cells (ATCC CCL-136) in DMEM supplemented with 2% FBS and 2 mM glutamine (at 37° C, 5% CO2 and 95% air). The virus was harvested from cell culture supernatant after removal of cells by centrifugation, and then quantified on RD cells using typical plaque assays. Subsequently, virus samples were inactivated under UV-light for 4-6 hours. Mock-infected samples were prepared in the same manner from cell culture supernatant but without the addition of EV71 virus. To demonstrate the virus binding capability of the AuNS-SCARB2 conjugates, 100 µL of 20 pM AuNS-SCARB2 conjugates were incubated at 4° C for 2 h with 100 µL of cell culture supernatant containing 107 pfu/ml of EV71. The AuNS-SCARB2 conjugates were also added to 100 µL of mock-infected cell culture supernatant as our negative control. During the incubation period, the absorption spectra of the mixtures were taken at 30 min intervals to determine the stability of the colloids. After incubation, the DH and Raman spectra of AuNS-SCARB2 in the biological media were obtained. The protocol was repeated at room temperature with a shorter incubation time of 15 min before obtaining the Raman spectra. To demonstrate specificity of


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

AuNS-SCARB2 to EV71, a cell culture supernatant sample containing 107 pfu/ml of dengue virus (DENV) was included as our second negative control.

Raman Spectra Acquisition and Analysis Raman spectra were acquired after incubating 100 µL of 20 pM AuNS-SCARB2 conjugates with 100 µL of controls or test samples for 15 min. All the Raman spectra in this study were acquired using the uRaman-785-Ci Raman spectroscopy system (Technospex Pte. Ltd., Singapore). The system utilized a 50 mW frequency stabilized laser at wavelength of 785 nm with linewidth approximately 100 MHz to excite the samples. This laser wavelength, which is far from the fluorescence excitation wavelength of biological samples, could minimize the generation of unwanted autofluorescence 28. The system was equipped with a TEC-cooled 2048 pixel CCD detector. The spectral resolution of the system was measured to be ~ 8.6 cm-1 and the spectra range span between 150 cm-1 and 2400 cm-1. All the spectra were obtained in a polystyrene (PS) cuvette (Bio-Rad Laboratories, USA) with an integration time of 60 s and were averaged from three measurements to minimize noise. The raw Raman spectra were processed using MATLAB to remove the autofluorescence background signal by third order polynomial curve fitting. In addition, the spectra were normalized to the 1,000 cm-1 peak of the cuvette to eliminate variations in the instrument’s sensitivity.

RESULTS AND DISCUSSION Synthesis and Characterization of AuNS-SCARB2 AuNS were synthesized by a SERS-optimized one-pot protocol using ascorbic acid as the reducing agent and silver nitrate as the shaping agent

25

. The synthesized AuNS had a DH of

105.12 ± 1.13 nm (Figure 2b) and a surface plasmon resonance (SPR) peak at 677 nm (Figure 2a,


10

Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


i). The SPR of AuNS was determined by a series of parameters including their size, shape and dielectric property of surrounding medium 29,30. In most applications, the SPR peak was typically near to the laser excitation wavelength for strong plasmon excitation and high local field enhancements

31

. While perfect matching would induce strong resonance effect, the SERS

enhancement would suffer from signal loss due to background absorption and presence of unwanted fluorescence. On the other hand, AuNS with a SPR slightly blue-shifted from the optical excitation of 785 nm has been reported to generate the strongest SERS enhancement in biological samples 32.

Figure 2. Characterization of synthesized AuNS and AuNS-SCARB2 at room temperature. (a) UV-Vis extinction spectra showing a red-shift of ≈ 20 nm in the SPR peak indicating the presence of SCARB2 on AuNS; (b) Histogram size distribution of the hydrodynamic diameter, DH from DLS of 50 pM AuNS and 20 pM AuNS-SCARB2 in H2O, showing an increase in DH of AuNS by ≈ 10 nm due to the SCARB2 corona around AuNS; (c) Transmission electron microscopy (TEM) images of AuNS dried on a silicon nitride TEM grid (2 µl of 50 pM AuNS,


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

spotted and dried 5 times); (d) Scanning electron microscopy (SEM) images of the same AuNS at 125,000X and (e) 275,000X magnification. Here, 5 µl of 20 pM AuNS was spotted on a silicon wafer and dried in vacuum to obtain the SEM images.

Multiple sharp spikes of synthesized AuNS were observed under TEM (Figure 2c) and SEM (Figure 2d and e), with an average spike density of 8-10 spikes per AuNS and each spike having an average length of ~20 nm as determined from the electron microscopic images (Figure 2c). Here, the size and morphology of AuNS exert a strong influence of the SERS enhancement. As SERS from plasmonic nanostructures originates predominantly from electromagnetic enhancement and to a lesser extent from electrochemical enhancement

33

, the sharp spikes on

AuNS were morphological features with a strong localized electromagnetic field and thus served as SERS “hot spots” 26. Studies have shown that AuNS exhibited a higher SERS enhancement in colloid compared to gold nanospheres or gold nanorods

26,34

. Therefore, we chose the AuNS

which we optimized from the one-pot synthesis protocol in our previous study to result in the strongest SERS enhancement

25

as the plasmonic NPs in this study.

In addition, the spiky surface of AuNS increased the surface area for non-covalent adsorption of SCARB2, thus allowing a larger SCARB2 payload on each AuNS. Here, the SCARB2 likely adsorbed on AuNS via a combination of electrostatic and hydrophobic interactions from the hydrophobic motifs on its sequence, or thiol bonds from cysteine amino acids in SCARB2. This passive adsorption was sufficiently stable in biological media and avoided the more complicated covalent binding in point-of-care probe preparations. From our protein titration of different SCARB2 concentrations on AuNS, we found an MPA of 40 µg/ml SCARB2 sufficient to protect 50 pM of AuNS from salt-induced aggregation (see Supporting Information, Figure S1).


12

Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The binding of SCARB2 on AuNS caused a 20 nm red shift in the peak absorbance of AuNS (Figure 2a, ii), which is typical of proteins adsorbed on surface of gold NPs 27. The average DH also increased to 115.1 nm (∆DH ~10 nm) (Figure 2b), which indicated successful conjugation of the SCARB2 proteins on AuNS. Interestingly, the size histogram distribution in Figure 2b shifted to the right collectively, without significant change in the size distribution profile. This indicated that the SCARB2 proteins coated the AuNS particles homogenously, and did not preferentially adsorb on any particular size population.

Raman Detection of EV71 We introduced the AuNS-SCARB2 to mock-infected cell culture supernatant without EV71 virus and observed the appearance of four distinguishable Raman peaks in the Raman spectrum of AuNS-SCARB2 at 390, 510, 670, and 910 cm-1 after 15 min at room temperature (Figure 3, iii). These four Raman peaks possessed intensities well above the fluctuation in the baseline signal of the Raman spectroscopy system and showed good repeatability across triplicates.


13

Page 14 of 37

910

670

510

4

(iv) + DENV

Raman Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

390


3 (iii) + Mock

2 (ii) + EV71

1 (i) AuNS-SCARB2

0 400

600 800 1000 Raman Shift (cm-1)

1200

Figure 3. Raman spectra of AuNS-SCARB2 added to mock-infected cell culture supernatant samples without EV71 (+Mock) showing the appearance of four Raman peaks at 390, 510, 670, and 910 cm-1 which was absent in the Raman spectrum of AuNS-SCARB2 in fresh DMEM (AuNS-SCARB2). Three of the peaks at 510, 670, and 910 cm-1 disappeared only in the presence of 107 pfu/ml EV71 virus (+EV71), but not in the presence of the same concentration of DENV virus (+DENV), showing the specificity of the peaks to EV71 virus. Measurements were


14

Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conducted after incubating 100 µL of 20 pM AuNS-SCARB2 with 100 µL of the test sample at 4° C for 2 h.

These four peaks were previously not present in the Raman spectrum of AuNS-SCARB2 in fresh DMEM (Figure 3, i). When the AuNS-SCARB2 was incubated in cell culture supernatant containing 107 pfu/ml EV71, three of the peaks at 510, 670, and 910 cm-1 disappeared, while the Raman peak at 390 cm-1 was diminished (Figure 3, red), leaving the Raman spectrum similar to that of AuNS-SCARB2 in DMEM alone. This change in the three Raman peaks intensity between EV71-infected and mock-infected samples (as our negative control) suggested that the three peaks at 510, 670, and 910 cm-1 could be potential markers for detecting EV71. To prove the specificity of EV71 detection, we also introduced AuNS-SCARB2 to cell culture supernatant sample containing 107 pfu/ml DENV, a different virus whose infection is mediated by different receptors (GAG, Mannose receptor, DC-SIGN, etc.) 35, with no affinity to SCARB2. In this case, the three peaks at 510, 670, and 910 cm-1 remained observable (Figure 3, iv), similar to that of mock-infected samples. This showed that the disappearance of the three Raman peaks was specific only to the presence of EV71. Previous studies on Raman spectrum of proteins largely attributed the peaks at 510, 670, and 910 cm-1 to disulfide bonds (-S-S-), carbon-sulfur bonds (-C-S-), and Cys and Tyr residues, respectively

18,36

. These moieties are strong Raman scatterers present in nearly all proteins

including the proteins present in cell culture supernatant in this study. Therefore, the appearance of these peaks in the spectrum of AuNS-SCARB2 in mock-infected samples but not AuNSSCARB2 in fresh DMEM media suggested that a protein-induced surface phenomenon on AuNS-SCARB2 was responsible for producing the SERS-enhanced Raman peaks.


15


Mechanism Behind Changes in Raman Peaks We sought to elucidate the mechanism behind the change in intensity of the three peaks at 510, 670, and 910 cm-1 by examining the colloidal stability of AuNS-SCARB2 from their UV-Vis spectra. The UV-Vis absorption (Figure 4a, DMEM) and the derived AI (Figure 4b, circles) of AuNS-SCARB2 in fresh DMEM did not change significantly with time. The mean DH of AuNSSCARB2 in the histogram size distribution was 115.1 nm (Figure 4c). This showed that AuNSSCARB2 retained its colloidal stability in biological medium in the absence of serum proteins.

A

0.6

0.4

(v) t = 2 h (iv) t = 1.5 h (iii) t = 1 h (ii) t = 0.5 h (i) t = 0

DMEM

0.8

0.6

0.4

0.2 500

600 700 800 Wavelength (nm)

B


C

900

0.8

0.6

500

+ EV71 + Mock 1.05


900

12

DMEM

Intensity (%)

Aggregation Index (AI)

1.10


0.4 500

900

+ EV71

1.0 Absorbance (a.u.)


Absorbance (a.u.)

+ Mock

0.8 Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

+ Mock + EV71 DMEM

8

4

1.00

0

30

60 Time (min)

90

120

0 10

100 1000 Hydrodynamic Diameter, DH (nm)

10000

Figure 4. Interaction of AuNS-SCARB2 with EV71 confers protection against aggregation of AuNS-SCARB2 in protein containing biological medium. (a) Time evolution of UV-Vis spectra of 20 pM 100 µL AuNS-SCARB2 when incubated with 100 µL fresh DMEM, mock-infected and EV71-infected cell culture supernatant containing proteins at 4° C for 2 h. (b) Time evolution of the AI of AuNS-SCARB2 derived from the UV-Vis absorption spectra, with the AI of each sample normalized to 1.00 at t = 0. (c) Histogram size distribution of the DH of AuNS-


16

Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SCARB2 in the three samples after incubation. Both UV-Vis spectra and change in DH indicated that AuNS-SCARB2 retained its colloidal stability in EV71-infected cell culture supernatant but aggregated in mock-infected cell culture supernatant.

When AuNS-SCABR2 was added to mock-infected cell culture supernatant, its absorbance peak at 700 nm decreased over time (Figure 4a, +Mock), suggesting aggregation of the AuNSSCARB2. This was confirmed by a gradual increase in its AI over time (Figure 4b, triangles) and a large shift in the histogram size distribution towards larger DH with an average DH of 490.2 nm (a 4.26-fold increase in the average DH of AuNS-SCARB2) (Figure 4c). The aggregation of AuNS-SCARB2 in mock-infected samples was likely induced by the proteins present in mockinfected cell culture supernatant since this was not observed in AuNS-SCARB2 in fresh DMEM media. Despite the larger average DH arising from aggregation, we did not observe any precipitation of the AuNS-SCARB2 during the SERS measurements. Unlike salt-induced aggregation which can be overcome by steric hindrance with a SCARB2 protein corona around AuNS 37,38, the same SCARB2 corona around AuNS may not be effective in conferring AuNS colloidal stability against protein-induced aggregation in mock-infected cell culture supernatant containing both serum and secreted proteins from cells. Here, the adsorption of these proteins on AuNS-SCARB2 may have caused misfolding and non-specific interactions between multiple proteins and AuNS-SCARB2 in the biological media formation of large AuNS aggregates

23,40,41

39

, triggering the

. Such protein-induced aggregation of NPs was

especially pronounced in biological media with high protein concentration such as blood, saliva, and the cell culture supernatant used in this study 42.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

The protein-induced aggregation of AuNS-SCARB2 in mock-infected cell culture supernatant was likely responsible for the appearance of the three Raman peaks at 510, 670, and 910 cm-1 since the aggregation of plasmonic NPs was known to significantly enhance the Raman spectrum of biomolecules localized within the inter-particle SERS “hot spots” formed from multiple particle junctions with intense electromagnetic field

12,25,43,44

. Thus, the aggregation of AuNS-

SCARB2 resulted in multiple SERS “hot spots” arising from these junctions

11

, leading to both

increased electromagnetic enhancement and increased charge transfer contribution that significantly enhanced the Raman signal of proteins within the SERS “hot spots”45. While there were a large number of proteins in the cell culture supernatant containing serum, including those secreted from cells, it would be nearly impossible to attribute the three peaks specifically to a particular protein or target. However, we suggest that the Raman peaks observed from aggregation of AuNS-SCARB2 in the mock-infected cell culture supernatant likely arose from Cys and Tyr residues of proteins localized in the SERS “hot spots” of the AuNS aggregates. Cys-rich proteins are widely-known to be prone to aggregation due to their tendency to form non-specific disulfide bonds with other Cys residues 46. Moreover, SCARB2 itself could also aggregate with other proteins since it possesses a Cys-rich motif on its luminal domain 47,48. The presence of this motif thus made AuNS-SCARB2 prone to non-specific interactions and aggregation with other proteins, thereby resulting in enhancement of Raman signals.

Anti-aggregation of AuNS-SCARB2 by EV71 Such an aggregation was not observed when AuNS-SCARB2 was introduced into cell culture supernatant containing 107 pfu/ml EV71 since its UV-Vis spectrum (Figure 4a, +EV71) and AI (Figure 4b, squares) remained relatively unchanged over time. The average DH increased only


18

Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


slightly to 148.2 nm (∆DH = 33.1 nm), with its histogram size distribution showing an increase much less than that of AuNS-SCARB2 without EV71 (Figure 4c). This increase in DH of 33.1 nm agreed with the typical size of EV71 virus 49, thus suggesting possible binding of EV71 to the surface of AuNS-SCARB2, conferring colloidal stability to AuNS-SCARB2 and preventing them from aggregating in the protein-rich cell culture supernatant. The absence of AuNSSCARB2 aggregation with EV71 present did not lead to the SERS enhancement of the three Raman peaks at 510, 670, and 910 cm-1 observed earlier without EV71. Here, we also noted that the binding of EV71 to AuNS-SCARB2 did not cause other appreciable change in the Raman spectra of AuNS-SCARB2. SERS is typically less sensitive to protein-protein interactions on the surface of the NPs as a highly concentrated protein solutions were typically required to detect molecular interactions on these NPs 50. Taken altogether, our observations show that the EV71 detection scheme was based on EV71induced anti-aggregation. When AuNS-SCARB2 was added to a negative sample, it formed large protein-NP aggregates that produced strong SERS signals caused by non-specific protein interactions between SCARB2 and other proteins in the cell culture supernatant. In the presence of EV71, however, the AuNS-SCARB2 was “protected” from these non-specific interactions as the specific binding of EV71 to SCARB2 minimized its interaction with other proteins in the cell culture supernatant. As a result, large protein-NP aggregates did not form with EV71, and the three Raman peaks were not observed. In the presence of 107 pfu/ml DENV virus, which was unable to bind and stabilize the AuNSSCARB2 from aggregating in protein-rich cell culture supernatant, the AuNS-SCARB2 aggregated and the three Raman peaks appeared (Figure 3, iv), similar to that of AuNS-SCARB2 in mock-infected sample. Interestingly, our two negative control samples (+Mock and +DENV)


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

could also be distinguished with other analytical techniques despite having similar Raman spectra. Using principal component analysis (PCA) on the Raman spectra, we were able to observe distinct clustering between the mock and DENV samples even with a limited sample size (Supporting Information, Figure S2). PCA is a non-parametric classification technique commonly used in Raman spectroscopy to discriminate samples with seemingly similar spectroscopic fingerprints. In our study, the distinction between +Mock and +DENV samples was probably due to the presence of DENV virus in the AuNS-SCARB2 aggregates that resulted in subtle differences in their Raman spectra. We would also like to highlight that such an anti-aggregation-based detection mechanism for EV71 was able to overcome the challenge of detecting targets in protein-rich samples commonly faced by other biomolecular detection assays, where the aggregation induced by protein-rich samples was considered a significant problem. In lateral flow assays, for example, non-specific aggregation induced by salivary proteins prevented gold NPs from flowing through the test membrane and compromised the accuracy of the assay

51

. Such an aggregation induced by

protein-rich samples was exploited in this study to generate the detection signals. Assays for detecting other biological modules based on this concept may also be developed with appropriate selection of capture ligands, as long as the sample is contained in a protein-rich biological media such as saliva 52 or plasma 53.

Comparison to UV-Vis Spectroscopy for Probing Aggregation Apart from SERS, aggregation of AuNS-SCARB2 could also be probed by colorimetric means based on their UV-Vis spectrum, which remained as one of the most common optical techniques for assays involving plasmonic NPs 27,37,49,54,55. Here, we compared both probing techniques after


20

Page 21 of 37

15 min of incubating AuNS-SCARB2 in various samples described earlier. While we obtained a higher mean AI from UV-Vis spectral measurements of mock samples without EV71 compared to samples with EV71 (Figure 5b), despite having very similar UV-Vis spectrum (Figure 5a), the difference was not statistically significant (Student’s t-test, p = 0.501).

A

B Absorbance (a.u.)

0.5 0.4 0.3

(ii) EV71 (i) Mock

2


(ii) EV71 (i) Mock

0.6

1

0

0.2 500

600

700

800

500

900

1000

C

1500 -1

Wavelength (nm)

Raman Shift (cm )

D Normalized Raman Intensity

1.0 Aggregation Index (AI)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.9

0.8

0.7 Mock

EV71

0.3

Mock EV71

* 0.2

* 0.1

0.0

-1

510 cm

-1

670 cm

-1

910 cm

Figure 5. Comparison of (a) UV-Vis spectrum and (b) Raman spectrum between (i) mock samples without EV71 and (ii) samples with EV71. (c) The AI derived from the UV-Vis spectra showed no significant difference between the two samples (two-tailed Student’s t-test, p = 0.501), while (d) significant differences between the two samples were observed in the Raman intensity of two (670 and 910 cm-1) out of three Raman peaks normalized to intensity at 1000 cm-1 (two-tailed Student’s t-test, p = 0.00024 and p = 0.056, respectively). Measurements were taken after incubating 100 µl of 20 pM AuNS-SCARB2 with 100 µL of mock and EV71 samples


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

for 15 min at room temperature. Error bars show the standard deviation for 3 different samples measured.

On the other hand, distinct differences in the Raman spectra, particularly at 670 and 910 cm-1 between the mock and EV71 samples were clearly observed (Figure 5b). The intensity of these two Raman peaks relative to a reference peak at 1000 cm-1 (due to the PS cuvette) from the mock sample without EV71 was significantly higher compared to that of the sample with EV71 virus (Student’s t-test, p = 0.00024 and p = 0.056 for 670 and 910 cm-1, respectively). Here, it was evident that in probing the aggregation of AuNS-SCARB2 for detecting the presence of EV71 in protein-rich samples, Raman measurements from colloidal SERS was a more sensitive technique than UV-Vis spectrum measurements, and hence a viable alternative to UV-Vis spectroscopy for developing aggregation-based detection assays.

Sensitivity of Raman-based Detection Scheme To determine the sensitivity of our anti-aggregation-based assay in detecting EV71, we introduced AuNS-SCARB2 to cell culture supernatant doped with varying concentrations of EV71 and observed no significant difference between the Raman spectra of the samples containing 105 and 106 pfu/ml of EV71 and the negative control of mock-infected cell culture supernatant without EV71 (Figure 6a). The peak intensity at 670 and 910 cm-1 relative to the reference peak at 1000 cm-1 remained relatively constant (Figure 6b and c, respectively), and decreased only at a higher concentration of 107 pfu/ml of EV71.


22

Page 23 of 37

B

510

670

910 (i) Mock

3

(ii) 10

670 cm-1 Normalized Raman Intensity

A

0.25 0.20 0.15 0.10 0.05 0.00 0

5 pfu/ml EV71

2

10 5 10 6 10 7 EV71 Concentration (pfu/ml)

(iii) 106 pfu/ml EV71

C

1 (iv) 10 7 pfu/ml EV71 0 400

600

800

1000

Raman Shift (cm-1)

1200

910 cm-1 Normalized Raman Intensity


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.10 0.08 0.06 0.04 0.02 0.00 0

10 5 10 6 10 7 EV71 Concentration (pfu/ml)

Figure 6. Concentration dependence of Raman peak heights. (a) Representative Raman spectra of AuNS-SCARB2 added to mock-infected cell culture supernatant samples (i) without EV71 and (ii – iv) cell culture supernatant with various concentrations of EV71. Raman peak intensity at (b) 670 cm-1 and (c) 910 cm-1 normalized to intensity at 1000 cm-1with varying concentrations of EV71. No significant change in the normalized Raman peak intensity at 670 and 910 cm-1 was observed at EV71 concentrations below 107 pfu/ml. Measurements were taken after incubating 100 µl of 20 pM AuNS-SCARB2 with 100 µL of mock cell culture supernatant and cell culture supernatant with varying concentrations of EV71 for 15 min at room temperature. Both peak intensities were normalized to the Raman peak at 1000 cm-1 from the PS cuvette. Error bars show the standard deviation for 3 different samples measured.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

This modest level of sensitivity was expected due to its anti-aggregation-based mechanism, where a minimum amount of virus particles was required to stabilize the AuNS-SCARB2 from protein-induced aggregation in order to distinguish between EV71-containing and EV71-free samples. Hence, the 20 pM of AuNS-SCARB2 used in this study would require 107 pfu/ml of EV71 to sufficiently bind and stabilize AuNS-SCARB2 from aggregating. Nonetheless, this detection limit was still comparable to other reported Raman-based detection assays for viruses which required 106 to 108 pfu/ml 13,15. Although the sensitivity of this assay could potentially be improved with a lower concentration of AuNS-SCARB2, which would require less EV71 virus particles to bind and prevent them from aggregation, a lower concentration of AuNS could also result in weaker SERS enhancement and thereby reduced sensitivity of this assay, based on our previously published work25. Hence, further studies to determine the optimum amount of AuNSSCARB2 added to the sample could maximize the sensitivity of this assay. Interestingly, the samples with 105 and 106 pfu/ml of EV71 showed slightly higher Raman intensities at 670 and 910 cm-1 compared to the sample without EV71 (Figure 6b-c). Apart from being insufficient to stabilize AuNS-SCARB2 against protein-induced aggregation, a lower amount of virus could also induce further aggregation. Since the EV71 capsid consists of several copies of viral proteins

56

, they present multiple possible binding sites for SCARB2 binding

57

.

Hence, several AuNS-SCARB2 could bind to a single EV71 particle under low concentrations of EV71 and cross-link to form aggregates of AuNS-SCARB2-EV71. These clusters could nonspecifically interact with other proteins in the biological medium to form even larger aggregates. This compounded aggregation effect could account for the slightly stronger Raman peaks in samples with lower amounts of EV71 (Figure 6b and c). In contrast, when sufficient amount of EV71 particles (i.e. 107 pfu/ml) were present, cross-linking by EV71 would be minimized as the


24

Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EV71 bound to AuNS-SCARB2 and formed a corona layer around it, therefore protecting the AuNS-SCARB2 from aggregation.

CONCLUSION The detection and analysis of biomolecules in protein-rich biological media such as saliva, serum or blood remains a challenge to many detection assays. Here, we described a novel antiaggregation detection scheme for detecting EV71 in protein-rich sample suitable for rapid diagnosis of EV71 virus-causing HFMD within 15 min and without the need for any temperature-controlled sample incubation. This approach involved introducing a bioconjugate (AuNS-SCARB2) comprising an EV71 capturing ligand to the sample, acquiring its Raman spectra after 15 min and analyzing the intensity of three Raman peaks at 510, 670 and 910 cm-1. The intensity of these peaks changed with the aggregation state of AuNS-SCARB2, which in turn depended on the presence of EV71. As such, this approach avoided the need for Raman reporters and complicated sample handling techniques. Despite its modest sensitivity comparable to other Raman-based virus detection scheme, this approach could be translated for point-of-care operations when combined with a handheld or portable Raman system. We envision that with further optimization to improve its sensitivity, such an approach could be used to detect any virus or protein in a protein-rich sample with appropriate selection of capture ligands.

ASSOCIATED CONTENT Supporting Information. Additional data on concentration titration to determine the minimum protecting amount (MPA) of SCARB2 on AuNS, and the principal components analysis (PCA)


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

of the Raman spectra for multiple EV71, DENV, and mock samples are available as Supplementary Information.

AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 1 Grant (R‐397‐000‐179‐112) . JJH Chu is funded by Ministry of Health (Singapore), NMRC Grant CBRG13nov023.

ACKNOWLEDGMENT S He would like to acknowledge the scholarship support from National University of Singapore Department of Biomedical Engineering. We also acknowledge the support from Barbara Poinard in acquiring the TEM images.

ABBREVIATIONS


26

Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AI, aggregation index; DENV, dengue virus; DLS, dynamic light scattering; EV71, Enterovirus 71; HFMD, hand, foot, and mouth disease; SCARB2, scavenger receptor class B, member 2; SERS, surface-enhanced Raman spectroscopy; TEM, transmission electron microscopy; UV-Vis, UV-visible absorption spectroscopy.

REFERENCES 1. Ang, L. W.; Tay, J.; Phoon, M. C.; Hsu, J. P.; Cutter, J.; James, L.; Goh, K. T.; Chow, V. T. PLoS One 2015, 10, e0127999. 2. Wang, Y.; Zou, G.; Xia, A.; Wang, X.; Cai, J.; Gao, Q.; Yuan, S.; He, G.; Zhang, S.; Zeng, M.; Altmeyer, R. Virol J. 2015, 12, 83. 3. Ling, B. P.; Jalilian, F. A.; Harmal, N. S.; Yubbu, P.; Sekawi, Z. Trop. Biomed. 2014, 31, 654-662. 4. Solomon, T.; Lewthwaite, P.; Perera, D.; Cardosa, M. J.; McMinn, P.; Ooi, M. H. Lancet Infect. Dis. 2010, 10, 778-790. 5. Mao, Q. Y.; Wang, Y.; Bian, L.; Xu, M.; Liang, Z. Expert Rev Vaccines 2016, 15, 599-606. 6. Fujimoto, T.; Chikahira, M.; Yoshida, S.; Ebira, H.; Hasegawa, A.; Totsuka, A.; Nishio, O. Microbiol. Immunol. 2002, 46, 621-627. 7. Huang, M.-L.; Chiang, P.-S.; Luo, S.-T.; Liou, G.-Y.; Lee, M.-S. J. Virol. Methods 2010, 165, 42-45. 8. Singh, S.; Chow, V. T.; Phoon, M.; Chan, K.; Poh, C. L. J. Clin. Microbiol. 2002, 40, 28232827. 9. Stiles, P. L.; Dieringer, J. a.; Shah, N. C.; Van Duyne, R. P. Annu. Rev. Anal. Chem. 2008, 1, 601-626. 10. Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 5935-5944. 11. Nie, S. Science 1997, 275, 1102-1106. 12. Xu, H.; Bjerneld, E. J.; Käll, M.; Börjesson, L. Phys. Rev. Lett. 1999, 83, 4357. 13. Shanmukh, S.; Jones, L.; Driskell, J.; Zhao, Y. P.; Dluhy, R.; Tripp, R. a. Nano Lett. 2006, 6, 2630-2636. 14. Driskell, J. D.; Shanmukh, S.; Liu, Y.-J.; Hennigan, S.; Jones, L.; Zhao, Y.-P.; Dluhy, R. A.; Krause, D. C.; Tripp, R. A. IEEE Sens. J. 2008, 8, 863-870. 15. Chang, C. W.; Liao, J. D.; Shiau, A. L.; Yao, C. K. Sens. Actuators, B 2011, 156, 471-478. 16. Xu, S.; Ji, X.; Xu, W.; Li, X.; Wang, L.; Bai, Y.; Zhao, B.; Ozaki, Y. Analyst 2004, 129, 6368. 17. Neng, J.; Harpster, M. H.; Wilson, W. C.; Johnson, P. a. Biosens. Bioelectron. 2013, 41, 316321. 18. Zhang, M. L.; Yi, C. Q.; Fan, X.; Peng, K. Q.; Wong, N. B.; Yang, M. S.; Zhang, R. Q.; Lee, S. T. Appl. Phys. Lett. 2008, 92.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

19. Harpster, M. H.; Zhang, H.; Sankara-Warrier, A. K.; Ray, B. H.; Ward, T. R.; Kollmar, J. P.; Carron, K. T.; Mecham, J. O.; Corcoran, R. C.; Wilson, W. C.; Johnson, P. A. Biosens. Bioelectron. 2009, 25, 674-681. 20. Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M. R.; Weigl, B. H. Nature 2006, 442, 412-418. 21. Cukalevski, R.; Ferreira, S. A.; Dunning, C. J.; Berggård, T.; Cedervall, T. Nano Res. 2015, 8, 2733-2743. 22. Moore, T. L.; Rodriguez-Lorenzo, L.; Hirsch, V.; Balog, S.; Urban, D.; Jud, C.; RothenRutishauser, B.; Lattuada, M.; Petri-Fink, A. Chem. Soc. Rev. 2015, 44, 6287-6305. 23. Zhang, D.; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Nano Lett. 2009, 9, 666-671. 24. Yamayoshi, S.; Yamashita, Y.; Li, J.; Hanagata, N.; Minowa, T.; Takemura, T.; Koike, S. Nat. Med. 2009, 15, 798-801. 25. He, S.; Kang, M. W. C.; Khan, F. J.; Tan, E. K. M.; Reyes, M. A.; Kah, J. C. Y. J. Opt. 2015, 17, 114013. 26. Nalbant Esenturk, E.; Hight Walker, a. R. J. Raman Spectrosc. 2009, 40, 86-91. 27. Yeo, E. L. L.; Chua, A. J. S.; Parthasarathy, K.; Yeo, H. Y.; Ng, M. L.; Kah, J. C. Y. RSC Adv. 2015, 5, 14982-14993. 28. Lee, C. H.; Hankus, M. E.; Tian, L.; Pellegrino, P. M.; Singamaneni, S. Anal. Chem. 2011, 83, 8953-8958. 29. Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.; Zhao, J.; Van Duyne, R. P. Nat. Mater. 2008, 7, 442-453. 30. Njoki, P. N.; Lim, I.-I. S.; Mott, D.; Park, H.-Y.; Khan, B.; Mishra, S.; Sujakumar, R.; Luo, J.; Zhong, C.-J. J. Phys. Chem. C 2007, 111, 14664-14669. 31. Kwon, Y. H.; Ossig, R.; Hubenthal, F.; Kronfeldt, H. D. J. Raman Spectrosc. 2012, 43, 13851391. 32. Yuan, H.; Fales, A. M.; Vo-Dinh, T. J. Am. Chem. Soc. 2012, 134, 11358-11361. 33. Khoury, C. G.; Vo-Dinh, T. J. Phys. Chem. C 2008, 112, 18849-18859. 34. Mehn, D.; Morasso, C.; Vanna, R.; Bedoni, M.; Prosperi, D.; Gramatica, F. Vib. Spectrosc 2013, 68, 45-50. 35. Cruz-Oliveira, C.; Freire, J. M.; Conceicao, T. M.; Higa, L. M.; Castanho, M. A.; Da Poian, A. T. FEMS Microbiol. Rev. 2015, 39, 155-170. 36. Rygula, A.; Majzner, K.; Marzec, K. M.; Kaczor, A.; Pilarczyk, M.; Baranska, M. J. Raman Spectrosc. 2013, 44, 1061-1076. 37. Ho, Y. T.; Poinard, B.; Yeo, E. L.; Kah, J. C. Analyst 2015, 140, 1026-1036. 38. Yang, H.; Heng, X.; Wang, W.; Hu, J.; Xu, W. RSC Adv. 2012, 2, 2671. 39. Kim, Y.; Ko, S. M.; Nam, J. M. Chem. Asian J. 2016, 11, 1869-1877. 40. Lacerda, S. H.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. ACS Nano 2010, 4, 365-379. 41. Dominguez-Medina, S.; Kisley, L.; Tauzin, L. J.; Hoggard, A.; Shuang, B.; AS, D. S. I.; Chen, S.; Wang, L. Y.; Derry, P. J.; Liopo, A.; Zubarev, E. R.; Landes, C. F.; Link, S. ACS Nano 2016, 10, 2103-2112. 42. Rausch, K.; Reuter, A.; Fischer, K.; Schmidt, M. Biomacromolecules 2010, 11, 2836-2839. 43. Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667.


28

Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


44. Kneipp, K.; Kneipp, H.; Kartha, V. B.; Manoharan, R.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. E 1998, 57, R6281. 45. Jana, N. R.; Pal, T. Adv. Mater. 2007, 19, 1761-1765. 46. Trivedi, M. V.; Laurence, J. S.; Siahaan, T. J. Curr. Protein Pept. Sci. 2009, 10, 614-625. 47. Holmes, R. S. J. Mol. Biochem. 2012, 1, 99-115. 48. Janssen, K.-P.; Rost, R.; Eichinger, L.; Schleicher, M. J. Biol. Chem. 2001, 276, 3889938910. 49. Liu, C.-C.; Guo, M.-S.; Lin, F. H.-Y.; Hsiao, K.-N.; Chang, K. H.-W.; Chou, A.-H.; Wang, Y.-C.; Chen, Y.-C.; Yang, C.-S.; Chong, P. C.-S. PLoS One 2011, 6, e20005. 50. Kengne-Momo, R. P.; Daniel, P.; Lagarde, F.; Jeyachandran, Y. L.; Pilard, J. F.; DurandThouand, M. J.; Thouand, G. Int. J. Spectrosc. 2012, 2012, 1-7. 51. Zhang, Y.; Bai, J.; Ying, J. Y. Lab Chip 2015, 15, 1465-1471. 52. Bonilla, C. A. J. Dent. Res. 1972, 51, 664-664. 53. Wadsworth, G. R.; Oliveiro, C. J. Br. Med. J. 1953, 2, 1138-1139. 54. Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14036-14039. 55. Thanh, N. T. K.; Rosenzweig, Z. Anal. Chem. 2002, 74, 1624-1628. 56. Plevka, P.; Perera, R.; Cardosa, J.; Kuhn, R. J.; Rossmann, M. G. Science 2012, 336, 1274. 57. Dang, M.; Wang, X.; Wang, Q.; Wang, Y.; Lin, J.; Sun, Y.; Li, X.; Zhang, L.; Lou, Z.; Wang, J.; Rao, Z. Protein Cell 2014, 5, 692-703.


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

TABLE OF CONTENTS GRAPHIC


30

Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44


Page 32 of 37

Page 33 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45


Page 34 of 37

Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Page 36 of 37

Page 37 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22



Exploiting the Anti-Aggregation of Gold Nanostars ... - ACS Publications

Recommend Documents