Organometallic-constructed Tip-based Dual Chemical Sensing by Tip

Subscriber access provided by UNIV OF LOUISIANA

Biological and Medical Applications of Materials and Interfaces

Organometallic-constructed Tip-based Dual Chemical Sensing by Tip-enhanced Raman Spectroscopy for Diabetes Detection Duo Lin, Yi-Cheng Lin, Shangwei Yang, Lan Zhou, Weng Kee Leong, Shangyuan Feng, and Kien Voon Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11950 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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 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 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.

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 24 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

ACS Applied Materials & Interfaces

Organometallic-constructed Chemical

Sensing

by

Tip-based

Dual

Tip-enhanced

Raman

Spectroscopy for Diabetes Detection Duo Lin, † ‖# Yi-Cheng Lin, ‡# Shang-Wei Yang, ‡ Lan Zhou, § Weng Kee Leong, ￡ ShangYuan Feng,†* Kien Voon Kong‡*

†Key

Laboratory of OptoElectronic Science and Technology for Medicine, Ministry of Education,

Fujian Provincial Key Laboratory for Photonics Technology, Fujian Normal University, Fuzhou, 350007, China. ‖

College of Integrated Traditional Chinese and Western Medicine, Fujian University of

Traditional Chinese Medicine, Fuzhou, 350122, China. ‡Department

§

of Chemistry, National Taiwan University, Taipei, 10617, Taiwan.

Department of Urology, Shanghai East Hospital, Tongji University School of Medicine.

Shanghai, 200000, China. ￡ Division

of Chemistry & Biological Chemistry, Nanyang Technological University, 639798,

Singapore.

KEYWORDS: Tip-enhanced Raman spectroscopy, Glucose, Thiol, Blood, Diabetes

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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 2 of 24

ABSTRACT. Tip-enhanced Raman spectroscopy (TERS) is capable of probing specific molecular information with high sensitivity, but dual chemical sensing remains a challenge. Another major hindrance to TERS chemical detection in biosamples such as blood is the interference from the strong absorptions of biomolecules. Herein, we report the preparation of an organometallic conjugate TERS tip. We demonstrate that organometallic chemistry can be perfectly coupled with TERS for dual-molecule sensing. The unique Raman signals generated by the organometallic compound circumvents signal interference from the biomolecules in blood, allowing the rapid analysis of two important molecules (glucose and thiol) in ultra-low volume (50 nL) samples. This enabled a correlation between thiol and glucose levels in the blood of nondiabetic and diabetic patients to be drawn.


2

Page 3 of 24 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



3


Page 4 of 24

1. INTRODUCTION Tip-enhanced Raman spectroscopy (TERS) is an ultra-sensitive detection method.1,2 The Raman signals of analytes can be dramatically increased, and its narrow line-widths and fluorescencequenching effects make it amenable for use in the analysis of biological samples.3-9 It has thus been widely used as a chemical sensor with low detection limits,10,11 as exemplified by its use in electrochemical studies,12-14 nucleobases/DNA analysis,15-18 and biomolecule detection.19 Raman spectroscopic techniques have also recently seen development in the field of bioorganometallic chemistry.20-22 In particular, a class of compounds known as metal carbonyl compounds have been used in biological immunoassays (metalloimmunoassays) including molecular therapy and imaging.23-31 The most recent application of these developments is the use of organometallic-conjugated nanoparticles for probing stroke biomarkers.22 Hydrogen peroxide (H2O2) and thiol-containing biomolecules are ubiquitous in nature and are vital in many biological processes. They are important for metabolic regulation,32,33 the maintenance of cellular redox potential,34 and the activity of many sulfhydryl enzymes.35 Deviation in the amount of thiols in blood can be a sign of health disorder. An elevated level of thiols has been associated with inflammation, myocardial infarction, and autoimmune diseases like rheumatoid arthritis.36,37 On the other hand, diabetic patients show significantly decreased levels of protein thiols in blood compared to healthy controls.38-41 This alteration in metabolism is due to hyperglycemia, which can significantly increase oxidative stress by non-enzymatic protein glycation, leading to increased free radicals and hence protein oxidation. Lower levels of thiols may thus correlate with an increased level of oxidation processes in diabetic subjects, as it is known that this disease condition can result in the oxidative modification of thiol molecules, leading to protein degradation and damage.


4

Page 5 of 24 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 correlation between thiols and glucose levels in blood is less well established. While glucose and thiols can currently be measured separately with electrochemical methods (e.g. glucose meter) and spectrophotometric assays (e.g. Ellman’s reagent), a method that can measure both glucose and thiols simultaneously is not known. A technique that can allow for such a simultaneous detection will certainly facilitate analysis time and, if it can be carried out with an ultra-low volume blood sample, potentially reduce pain associated with blood collection. TERS has recently been demonstrated for imaging single-walled carbon nanotubes in a liquid environment.42 A novel TERS tip for application in biology and biomedicine has also been constructed.43 We present here a novel procedure for the detection of glucose and thiols in a liquid environment with TERS, based on the use of the CO stretching vibrations of a TERS tip construct.

2. EXPERIMENTTAL SECTION 2.1 General procedure The triosmium carbonyl cluster Os3(CO)10(μ-H)2, was prepared according to the reported procedure.44 Os3(CO)12 was purchased from Oxkem. HRMS were measured using ESI mode on a Waters UPLC-Q-Tof MS mass spectrometer. The Raman spectra were detected by a Raman microspectrometer (Renishaw InVia) with a Peltier cooled charge-coupled device camera and a 785 nm diode laser. The laser power at the surface of sample was about 6.2 mW and the acquisition time was 10s with a long distance 50× objective lens. The software package WiRE 4.3 was employed for spectral processing, including polynomial multipoint and curve fitting. Prior to each measurement, a silicon wafer with a Raman signal at 520 cm-1 was used for instrumental calibration. For each sample, five TERS spectra were obtained. For quantification, peak heights


5


Page 6 of 24

were used instead of intensity in order to avoid problems associated with overlap of the thiol and glucose peaks.

2.2 TERS experiments TERS measurements were made on an AFM system (nanonics MV 2000) and a Raman microscope (Renishaw) equipped with a 785 nm laser. The TERS tip was purchased from a supplier and calibrated using silicon as test object. A gold nanoparticle 150 nm in diameter was attached to the apex of a transparent glass cantilever.19,45,46 A long distance 50× objective lens (Olympus) was used. The TERS tip was positioned to a liquid cell with AFM controller boxes provided by Nanonics, and the laser beam was focused at the tip. An ethanolic solution (500 μL) of Os3(CO)10(μ-H)2 (10 mM) was added into the liquid cell to sufficiently cover the apex of the TERS tip, and this was incubated for 1 h. Functionalization of the tip with Os3(CO)10(μ-H)2 was confirmed by acquiring a TERS spectrum from the apex of the tip. TERS spectra of the thiol sample/blood samples (50 nL) were collected using the HC-PCF fiber (NKT Photonics). The side channel of this fiber segment contains the maximum volume of ~50 nL.47,48 The sample in the fiber was added into the container with the Os3(CO)10(μ-H)2 solution above and incubated for 10 minutes before the TERS spectrum was collected. A similar procedure was used for glucose detection except for the further addition of an aqueous solution (50 nL) of glucose oxidase (GOx; 1mg/mL) and incubation (30 min), before the TERS measurement. The tip was cleaned after each experiment by sonication in absolute ethanol at low frequency (ELMA ultrasonic cleaner P120H) for 10 mins. A reference spectrum from the silicon was recorded to confirm removal of contaminants.


6

Page 7 of 24 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 calibration curve was fitted by linear regression, and the sensitivity measured as the slope. The limit of detection (LOD) was obtained as the standard deviation from multiple measurements and calculated as: LOD = 3.3× (standard deviation / slope of calibration curve).49-52

2.3 Computational studies The computational studies were performed with DFT theory using Becke’s three parameter hybrid function, and the Perdew-Wang’s gradient-corrected correlation function (B3PW91), together with the LanL2DZ basis set. Vibrational frequencies were computed at 298.15 K and 1 atm pressure. Optimized geometries were characterized by all real frequencies. The Gaussian 09 suite of programs was used in all these calculations.

2.4 Detection of glucose and thiol in clinical blood samples Clinical blood samples were collected and stored immediately at −20 °C before use. The procedure of sample collection was approved by the local ethics committee (Medicine and Life Science Department, Tongji University: 2012-DF-01), and informed consents were given by all patients. For verification, the glucose concentration was also directly measured with a glucose meter with a sample volume of 5 μL. For thiol detection, a DTNB commercial kit was used (Abcam). The blood sample was diluted 50-fold with water before assay. The sample (50 μL) was placed into a 96-well plate, the thiol probe (50 μL) added, and then incubated for 5 min before reading the plate at 305 nm.


7


Page 8 of 24

3. RESULTS AND DISCUSSION There have been numerous reports that described the interaction of gold nanoparticles with H2O2 to generate hydroxyl radicals.53-55 H2O2 adsorbed onto the gold nanoparticle surface causes partial electron transfer between them.53 This leads to a positively charged gold surface, which may induce a shift in the CO vibrational frequencies of an organometallic conjugated onto a gold nanoparticle at the apex region of a TERS tip. The precursor compound, Os3(CO)10(μ-H)2, which we have chosen for the construction of the H2O2-responsive TERS tip, is an electronically unsaturated (46 electrons) osmium carbonyl cluster (Figure 1).56 This cluster has a high affinity towards the thiol (SH) functionality.57,58, A procedure that involves dipping a TERS tip into a solution of Os3(CO)10(μ-H)2 followed by addition of a GOx-treated blood sample for the dual detection of glucose and thiol can thus be envisaged. The spectrum from a TERS tip treated in a solution of Os3(CO)10(μ-H)2 followed by the addition of cysteine shows two sharp peaks at 2019 and 2111 cm-1 (Figures 1A and B). The high frequency band (2111 cm-1) could be assigned to A ˊ mode, whereas the low frequency band (2019 cm-1) corresponds to A˝ mode.59,60 The spectrum from the TERS tip after treatment with Os3(CO)10(μ-H)2 shows a sharp peak at 2019 cm-1 (Figure 1C). This is the same as that obtained from the reaction of Os3(CO)10(μ-H)2 with gold nanoparticles which we have reported earlier,20 and hence can be attributed to the formation of an Os3(CO)10(μ-H)(μ-Au) species. On the other hand, the spectrum from an unmodified TERS tip dipped into the solution mixture above shows a very sharp peak at 2111 cm-1 and a broad peak at 2004 cm-1 (Figure 1C); this can be attributed to the formation of Os3(CO)10(μ-H)(μ-S-cysteine), and is corroborated by the mass spectrum (Figure S1). The 2111 and 2004 cm-1 bands can similarly be attributed to the A ˊ and A˝ modes, respectively, in accordance with the Cs point symmetry of Os3(μ-H)(CO)10(μ-S-cysteine).


8

Page 9 of 24 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 formation of Os3(CO)10(μ-H)(μ-Au) from the interaction of Os3(CO)10(μ-H)2 with gold has been determined by Li, et. al., using TOF-SIMS.61,62 The possibility of oxygen of the CO ligand of Os3 fragment binding to gold (Os-C≡O-Au) is unlikely. The HOMO (3*) and LUMO (2*) of CO are localized mainly at the carbon atom and are already involved in bonding interactions (-donation and -back donation, respectively) with the Os atom. Any further interaction with Au would require further donation from the HOMO or from the lower-lying pair of C-O  bonds. When the laser is directed away from the TERS tip, it shows no detectable CO signal (Figure 1D (I)); signals were only observed when the laser was focused on the TERS tip (Figure 1D (II)). There was an increase in the CO intensity by a factor of ~8000, estimated as the ratio of the signal intensity of the TERS measurement to that of a Raman spectrum of 50 mM solution of the osmium carbonyl cluster. In the mixture of Os3(CO)10(μ-H)2 and cysteine, a weak cysteine signal was observed and strong signals of cysteine and Os3(CO)10(μ-H)(μ-S-cysteine) were again detected only when the laser was focused on the TERS tip (Figure 1D (III-IV) sharp peak at 683 cm-1 can be assigned to C–S stretching band.49)


9


Page 10 of 24

Figure 1. (A) Reaction scheme for Os3(CO)10(μ-H)2 with cysteine and TERS tip. The short line extending from Os represents a coordinative bond to carbon monoxide (Os–CO). (B) Spectrum from TERS tip after incubation with a solution of Os3(CO)10(μ-H)2 and cysteine. (C) Overlap of TERS spectra for Os3(CO)10(μ-H)(μ-Au) and Os3(CO)10(μ-H)(μ-S-cysteine). (D) Spectra of Os3(CO)10(μ-H)2, Os3(CO)10(μ-H)(μ-Au), cysteine. A similar spectrum was observed if cysteine was replaced with glutathione (GSH), or Cor N-protected cysteine (Figures S2-S5). On the other hand, TERS experiments performed with


10

Page 11 of 24 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


various amino acids show that the peak at 2111 cm-1 was not observed with thiol-free amino acids (Figure 2A); only the peak assignable to Os3(CO)10(μ-H)(μ-Au) was observed. For cysteine, the intensity of the 2111 cm-1 peak increased with concentration, with the LOD estimated at 0.16 mmol/L (Figure 2B and Figure S6). Since the two peaks do not overlap, we now have two independent markers in one system. Addition of H2O2 to an Os3(CO)10(μ-H)(μ-Au)-conjugated TERS tip led to a blue shift of the CO peak (Figure 3A). The TERS spectrum of Os3(CO)12 did not exhibit any shift upon treatment with H2O2 (Figure S8), confirming that H2O2 did not react directly with the triosmium cluster. It has been reported that the surface potential of gold nanoparticles can be affected by small adsorbates, and that this can result in vibrational frequency shifts of molecules that are attached onto the gold surface. It is thus plausible that a similar shift occurred with the Os3(CO)10(μ-H)(μAu)-conjugated TERS tip.63-65 This shift can be rationalized on the basis of the metal-CO bonding, which comprises a metal-to-CO  donation and a CO-to-metal  back-donation. The former interaction involves electron-donation from a slightly antibonding * orbital, and the latter involves electron-donation into a pair of antibonding * orbitals, of CO. A positive potential on the nanoparticle surface, for example, would induce a positive charge on the osmium atom, which would in turn lead to an increase in  donation and a decrease in  back-donation. Both changes would result in an increase in the CO bond order and hence a shift to a higher frequency for the CO bond vibration.


11


Page 12 of 24

Figure 2. (A) TERS spectrum of Os3(CO)10(μ-H)2 treated with various amino acids. (B) Plot of TERS νCO Raman intensity versus concentration of cysteine. The peak shift was also correlated with the concentration of H2O2 (Figure 3B), but not with the pH (from 4 to 12) (Figure S9). These observations are again consistent with the suggestion above of a positive potential on the gold surface inducing a positive charge on the osmium atom. This was also corroborated by a density-functional theory calculation using Os3(CO)10(μ-H)(μAu) as the model, which showed that the Raman spectrum for the CO vibrations of the neutral species would shift to higher wavenumbers in the charged species [Os3(CO)10(μ-H)(μ-Au)]+ (Figure 3C). The sensitivity of the Os3(CO)10(μ-H)(μ-Au) conjugate to H2O2 concentration suggests that it may form the basis for the determination of glucose via an enzymatic assay. Glucose oxidase (GOx) is an enzyme that converts glucose to gluconic acid and H2O2.66 To do this, an Os3(CO)10(μH)(μ-Au)-conjugated TERS tip was immersed into a sample of glucose mixed with GOx, with the excitation laser focused onto the apex of the tip. A plot of the TERS peak for the CO vibration


12

Page 13 of 24 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


against the concentration of glucose showed that the LOD was 0.21 mmol/L (Figure 4B and Figure S10). As summarized in Figure S6 and S10, good consistency in the measurements pointed to success of this platform in the bioassay. Control experiments using galactose, maltose and fructose (5 mM) showed no observable peak shifts (Figure 4A), thus demonstrating selectivity for glucose. The simultaneous determination of glucose and thiol at a single spot was also demonstrated (Figure 4C). For the glucose detection, a significant shift in the CO peak from 2019 cm-1 to 2031 cm-1 was observed upon the addition of glucose into the GOx solution. This is due to the generation of H2O2 from glucose by GOx (glucose + GOx → gluconic acid and H2O2). The peak shift continues and the intensity increases as glucose concentration increases (Table S1). Similarly, the intensity of the peak at 2111 cm-1 increases as the concentration of thiol increases. Repeated use of the GOx, however, eventually leads to smaller peak shifts (from 2019 cm-1 to 2024 cm-1) as a result of degradation of the GOx by H2O2,67,68 thus fresh GOx has to be used for each new experiment. The platform was further verified with samples spiked with a known amount of thiol or glucose; acceptable precision was obtained (Table S2).


13


Page 14 of 24

Figure 3. (A) TERS spectra of Os3(CO)10(μ-H)(μ-Au)-conjugated TERS tip with (bottom) and without (top) addition of H2O2. (B) TERS responses of Os3(CO)10(μ-H)(μ-Au)-conjugated TERS tip to varying concentrations of H2O2. (C) Computationally optimized structure of Os3(CO)10(μH)(μ-Au), and the calculated Raman spectra (νCO) for neutral Os3(CO)10(μ-H)(μ-Au) and [Os3(CO)10(μ-H)(μ-Au)]+.


14

Page 15 of 24 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


Figure 4. (A) TERS spectra of Os3(CO)10(μ-Au)2-conjugated TERS tip with galactose, maltose, fructose and glucose. (B) Plot of TERS CO stretching frequency versus concentration of glucose. (C) TERS dual sensing for the simultaneous detection of different concentrations of thiol and H2O2. The concentration of thiol and glucose in a very low volume (50 nL) of human blood samples was also determined, using a capillary needle to collect the samples for TERS measurement (Figure S11).47,48 The blood samples were from healthy controls and diabetic patients, and were collected after fasting. The diabetic patients were those with the condition for more than two years, and patients on antioxidant medication were excluded. Healthy controls were not on medication. As shown in Figure 5A, blood samples show typically strong signals in the 100-1800 cm-1 region, which can interfere with direct glucose or thiol detection. This is obviated with the CO signal from the Os3(CO)10(μ-H)(μ-Au)-conjugated TERS tip, thus enabling an accurate and sensitive detection. The thiol level in blood was significantly lower in diabetic


15


Page 16 of 24

patients compared to that of healthy patients (Figure 5B), due to the increase of oxidation processes such as hyperglycemia, which can increase the level of radicals through glucose auto-oxidation.69

Figure 5. (A) TERS spectrum after incubated with blood samples. (B) Detection (mean ± SD.) of glucose and thiol from blood. (One-way Anova followed by Tukey test; * = p

Organometallic-constructed Tip-based Dual Chemical Sensing by Tip

Recommend Documents