Novel resonance light scattering testing system for human-thrombin using gold nanoparticle modified aptamers in sandwich manner

Chenxiao, Li Zhengping, Liu Zhuo
(Chemistry department, Hebei university, Baoding 071002;Academic administration, Shijiazhuang University, Shijiazhuang 050035, China)

Abstract A novel nanoparticle-based detection of human -thrombin, based on resonance light scattering (RLS), is described. In this protocol, two DNA aptamers that recognize different position of thrombin were employed. To obtain signal, both of the aptamers were alkanethiol modified and bound to gold nanoparticles (GNPs). When each aptamer connected to separate exosite of human-thrombin in a sandwich manner, GNPs can self-assemble to form a network structure, which resulted in greatly enhanced resonance light scattering (RLS)This new method recognized its target protein with high specificity and high sensitivity in homogenous solution (detection limit (3)0.72 nM).

1 Instruction
Aptamers are single-stranded oligonucleotides that can selectively bind to amino acids, drugs, proteins and other molecules. These aptamers, have low-nanomolar dissociation constants for large targets such as proteins and micromolar dissociation constants for smaller molecule targets. Aptamers are selected using a relatively rapid in vitro selection process known as systematic evolution ligands by exponential enrichment (SELEX) process1-4. Generally, SELEX-derived aptamers could be considered as functional analogues of monoclonal antibodies, and it has many advantages to antibody5-9, such as their facile in vitro synthesis, cheep cost, thermal stability, easy modification and high purity. Therefore aptamers are emerging as an ideal class of molecules that can rival commonly used antibodies intarget protein assays. Recently, several analysis methods using aptamers have been reported including fluorescence 10-14, chemiluinescence15, colorimetric16-18, electrochemistry 19,20, AFM21 and so on. Most of them are fluorescence assay, for example, resonance fluorescence energy transfer (FRET). However, these kinds of method need to make clear of the precise binding sites and conformational changes of the aptamer before designing the aptamer probes. Recently, Valeri Pavlov designed a colorimetric method base on gold nanoparticles to detect thrombin in homogeneous and heterogeneous respectively18, it can detect 2 nM thrombin, but the process of it is too complicated for the application in protein detection.
Herein, we designed a model system based on self-assembly of GNPs to signal aptamer/protein binding using resonance light scattering (RLS) technique and colorimetric method. We use human-thrombin and its two aptamers for the construction of a model sandwich system. For protein detection, GNPs are modified with two aptamers, each of whom is specific to different position of human α-thrombin. In the presence of human-thrombin, these modified GNPs can self-assemble to form a network structure, the interparticle cross-linking and surface plasmon coupling between GNPs leads to a significant RLS signal increasing. Therefore, we explored a simple and sensitive RLS method using GNPs to sensing a certain protein.

2 Experimental
Tetrachloroauric acid (HAuCl·4HO) was obtained from Sinopharm Group Chemical Reagent Co., Ltd reagent Inc. Human-thrombin (all of the thrombin mentioned below is human-thrombin) was obtain from Haematologic Technologies, Inc. 3-alkanethiol-modified aptamers having the sequence 5- AGT CCG TGG TAG GGC AGG TTG GGG TGA CTT TTT TTT TTT T-SH 3′ and 5- TGG TTG GTG TGG TTG GGT GCT TTT TTT TTT TT-SH 3′ were purchased from Takara Biotechnology (Dalian) Co., Ltd. Albumin bovine serum (BSA), albumin human serum (HSA) , insulin(from bovine pancreas) and-Globulins were all purchased from sigma. All other reagents were of analytical reagent grade and used as purchased without further purification. Doubly distilled water was used throughout.
2.2 Apparatus
RLS intensity and RLS spectra were measured with a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan), equipped with a 150 W high pressure Xenon lamp. The absorption spectra were obtained by a TV-1901 UV-VIS spectrophotometer (Beijing Purkinje General Instrument Co., Ltd, Beijing, China). The TEM images of the gold nanoparticles were acquired on a JEM-100SX transmission electron microscope (Tokyo, Japan). A GL-16G-Ⅱhigh speed refrigerated centrifuge (Shanghai Anting Scientific Instrument Co., LTD, Shanghai China) was used for the centrifugation of gold nanoparticles solution. A WH-861 vortex mixer (Taicang Instrumental Co., Jiangsu, China) was used to blend the solution.
2.3 Preparation
2.3.1 Preparation of Gold Nanoparticles

GNPs were prepared by citrate reduction of HAuCl22, 23. All glassware used in the preparation was thoroughly cleaned with chromate washings (cleansing solution), rinsed in water, and oven-dried prior to use. A 100-mL of 0.01% HAuCl solution was heated to boil, and then 3.5 mL of the 1.0% trisodium citrate solution was quickly added with vigorous stirring. Boiling was continued for another 6 min. The solution naturally cooled to room temperature after moving away from the heater, and then stored at 4℃
2..3.2 Preparation of Alkanethiol Oligonucleotide-modified Gold Nanoparticles
GNPs were modified with alkanethiol oligonucleotides by adding alkanethiol oligonucleotides to 0.5 mL aqueous GNP solution to a final oligonucleotide concentration of 1M. After being incubated for 24 h at room temperature, the solution was adjusted to the pH value and ionic strength of the phosphate buffer (10 mM, pH 7.0) and allowed to stand for 8 h. Then 2 M NaCl was added to the solution to bring the total NaCl concentration of the solution to 0.05 M. This was repeated 10 h later by adjusting the NaCl concentration to 0.1 M. The solution was allowed to “age” under these conditions for additional 30 h. Excess oligonucleotides were then removed by centrifugation for 25 min at 12000 rpm. Following removal of the supernatant, the red oily precipitate was washed once with 0.1 M NaCl, 10 mM phosphate buffer (pH 7.0) by successive centrifugation. Finally, the red oily precipitate was redispersed in 0.5 mL doubly distilled water and stored at 4℃. We donate the concentration of the resulting solution of aptamer-modified GNPs to be 1×. Additionally, in the colorimetric method, we obtained a concentrated aptamer-modified GNP solution by redispersed the precipitate in 0.25 mL doubly distilled water in the last step.
2.5 Assay
Standard Procedure of RLS Detection.In a 0.5 mL centrifuge tube, 30L NaHPO-NaHPO buffer (pH=7.4, 0.2 M), 10L 2 M NaCl, 30L 50 mM KCl, and 11L each kind of aptamer-modified GNPs (final concentration was 0.073×) were diluted to 300L with water and then mixed with a vortex mixer. In succession, added a certain volume of thrombin and pipeting to blend thoroughly. After incubation 30 minat room temperature, RLS spectra were obtained with simultaneous scanning with the same excitation and emission wavelengths from 200 to 700 nm by F-4500 fluorescence spectrophotometer and the RLS intensities were measured at the wavelength of 550 nm. The slit width and PMT voltage of the measurements were 5 nm and 400 V, respectively.

3 Results and discussion
3.1 RLS method
3.1.1 Spectral characteristics
The principle of the sandwich system for the detection of thrombin using aptamer-modified GNPs as probes is depicted in scheme 1. Thrombin act as a bridge to pull the GNPs hand in hand, and the distance among crosslinked GNPs is less than the average diameter of them, which result in the RLS signal enhancement and color change. Figure 1 shows the RLS spectral features of thrombin, aptamer-modified GNPs and the complex of them. The thrombin has weak RLS signals over the wavelength range 200-700 nm, and the modified GNPs exhibit two low light scattering peaks located at 300nm and 530nm respectively. However, when modified GNPs mix with thrombin, a wide RLS band in 300-500 nm and a very sharp peak at 550nm can be observed, which indicate an interaction between modified GNPs and thrombin has been occurred.
Figure RLS spectra of (a) 8 nM thrombin; (b) 0.073× aptamer-modified GNPs; (c) mixture of (a) and (b). Experimental condition: 0.02 M NaHPO-NaHPO buffer pH=7.4; NaCl concentration: 67 mM;

Scheme 1. Schematic representation of aggregation of aptamer-modified GNPs in the presence of Human α-thrombin

The diameter of GNPs is approximately 16 nm, which is less than 1/20 of incident light wavelength. Therefore, the light scattering of GNPs can be considered as Rayleigh scattering. According to the Rayleigh law, if the spectral characteristics (such as the convolution of the lamp spectrum, the transmission of the monochromators, and the spectral response of the photomutiplier tube) of the spectrofluorometer are corrected, the intensity of the light scattering should be proportional to the square of the scatters volume and inversely proportional to the quartic of the incident light wavelength. However, if using an uncorrected spectrofluorometer equipped with a high-pressure Xenon lamp, the Rayleigh light scattering spectrum of aqueous solution generally has a maximum light scattering peak at about 300 nm and gradually decreases with the incident light wavelength increasing24
According to the RLS theory24, RLS effect is observed and increased scattering intensity at or very near the wavelength of absorption of the aggregated molecular species. The effect can be dramatically enhanced when strong electronic coupling exists among the chromophores. GNPs are the aggregates of Au atoms at nanoscale dimensions and their absorption peak at 520 nm. Therefore, the light scattering peak of GNPs at 550nm can be considered as RLS peak. When thrombin is added to the GNP solution, the GNP aggregate and the interparticle distance in these aggregates decreases to less than the average particle diameter, which results in the shift of absorption band to longer wavelength 545 nm, as a result of electric dipole-dipole interaction and coupling between the plasmon of neighboring particles in the formed aggregates. As shown in Figure 2, when thrombin added, the absorption peak of modified GNPs shifts from 520nm to 545 nm.
Figrue 2. UV-Vis absorption spectra of (a) 0.1× aptamer-modified GNPs; (b) mixture of 0.1× aptamer-modified GNPs and 2.4 ×10-8 M thrombin. The experimental condition was the same as Figure 1.

   Therefore, it can be concluded that the size increasing of scattering particles can produce Rayleigh light scattering enhancement in the wavelength range from 200-700 nm, and meanwhile the electronic coupling of GNPs originated form the interaction among GNPs can greatly enhance RLS at 550 nm.
3.1.2 Effect of the pH and salt concentration
pH value was first investigated. As shown in Figure 3, pH has slim effect on the RLS signal, so we chose the physiological pH value 7.4 as the optimum pH. Salt concentration was also studied. It was found that the optimal salt concentration was 67 mM(Figure 4).
Figure 3. Effect of pH on RLS. (a)Blank (aptamer-modifed GNPs); (b) sample (mixture of mixture of the aptamer-modified GNPs and thrombin); Experimental condition: NaCl concentration: 67mM; aptamer-modified GNPs concentration:0.073
Figure 4. Effect of NaCl concentration on RLS signal. (a)Blanks(aptamer-modified GNPs); (b)samples (mixture of the aptamer-modified GNPs and thrombin). The blanks were treated in the same way as the samples without thrombin. Experimental condition:0.02 M NaHPO-NaHPOpH=7.4, aptamer-modified GNP concentration: 0.073×

3.1.3 Effect of GNP concentration on the stability of RLS
GNP concentration has effect on the stability of the RLS signal. We can see from Figure 6 that as the concentration of GNPs increasing, the RLS signal reached its peak value quickly and decreased quickly too. To obtain steady signal, we had to choose a relatively low GNP concentration, as shown in Figure 5, when GNPs concentration was 0.073×, the signal can kept relative stable after it reach its peak value, so we select 0.073× as the work concentration.
Figure 5. Effect of aptamer-modified GNPs on the stability of RLS signal. Aptamer-modified concentration:(a) 0.23×; (b) 0.16×; (c) 0.11×; (d) 0.093×; (e) 0.073×;Experimental condition: 0.02 M NaHPO-NaHPO pH=7.4; NaCl concentration: 67 mM.
Figure 6. Standard curve with thrombin. Every data was measured in triplicate.

3.1.4 RLS assay for thrombin
Under the optimized experimental condition mentioned above, the relationship between RLS signal and the concentration of thrombin was investigated. Figure 6 shows the RLS intensity versus the concentration of thrombin, the detection limit ( 3
σ) was 0.72 nM, but the linear range is very narrow ( 2 nM-5.6 nM ), which may due to the low GNP concentration. So, we tried to find a new method to obtain broad linear range.


3.1.5 Interference study of RLS
To validate the specificity of the RLS method, we add foreign proteins with fixed amount of thrombin. We can see from Table. 1 other proteins whose concentrations are thousands or hundred times higher than that of thrombin cannot affect the detection of thrombin. This indicates that this method is highly selective.

Table 1.Interference of different proteins

Protein Concentration RSD(%)
BSA μ 4.7
HSA μ -1.8
Insulin μ -1.3
γ-Globulins μ 2.9

The concentration of thrombin was 8 nM.

4 Conclusions
Nanoparticle-based detection system for thrombin using the two different aptamers in sandwich manner was developed. Using RLS method, nM of the lower detection limit was obtained, and none of the other protein used can cause significant RLS signal enhancement. Otherwise, it would not be so difficult to obtain two aptamers recognizing different part of the target protein, so the nanoparticle-based detection system can be easily applied to detecting other kinds of proteins. The work illustrate the feasibility of detection system for proteins using GNPs modified aptamers as probes, which would be a key technology for other kinds of proteins.