Label-Free and Sensitive Detection of Thrombomodulin, a Marker of

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Label-free and sensitive detection of thrombomodulin, a marker of endothelial cell injury, using quartz crystal microbalance Yiqun Luo, Tong Liu, Jiaming Zhu, Liyan Kong, Wen Wang, and Liang Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02447 • Publication Date (Web): 28 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Analytical Chemistry

Label-free and sensitive detection of thrombomodulin, a marker of endothelial cell injury, using quartz crystal microbalance Yiqun Luo, Tong Liu, Jiaming, Zhu, Liyan Kong, Wen Wang, Liang Tan* Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, PR China

ACS Paragon Plus Environment

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ABSTRACT: Thrombomodulin (TM), an integral glycoprotein on the surface of endothelial cells, can be released during endothelial cell injury and the levels of serum TM are regarded as an important parameter of activity in vasculitides in vivo. Quantitative detection of TM and investigation on the release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells using quartz crystal microbalance (QCM) were achieved in this work. Anti-antibody (AAb) and bovine serum albumin (BSA) were bound on gold nanoparticles (GNPs) to construct BSAGNPs-AAb nanocomposites and they were characterized by transmission electron microscope, UV–vis and infrared spectrophotometry, respectively. The capture of the nanocomposites on the TM antibody modified electrode, which was testified by scanning electron microscope, could result in a great decrease of the resonant frequency (f0). This binding was effectively inhibited by the beforehand immobilized TM proteins on the electrode surface due to the strong steric hindrance effect. It led to the decrease of the frequency changing extent. The relative frequencyshift was found to be proportional to the logarithm of the TM concentration from 10 to 5000 ng mL-1 with a detection limit of 2 ng mL-1. By analyzing the growth medium used for cell incubation, the release of sTM by the injured HUVEC-C cells in the presence of H2O2 was confirmed. The sTM amount in the growth medium was increased with the enhancement of contact time of the cells with H2O2, proving that sTM may serve as a specific marker of endothelial cell injury.

Keywords: thrombomodulin, vascular endothelial cells, quartz crystal microbalance, gold nanocomposites, label-free detection


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INTRODUCTION Blood vessel walls form a selective barrier for the transport of molecules between blood and tissues. Endothelium constitutes the largest homogenous surface of body for actively mediating the immune defense. It is composed by squamous and flattened epithelial cells that form a thin layer on the interior surfaces of all blood vessels and lymph vessels through the entire circulatory system. Endothelial cells (ECs) in direct contact with blood are called vascular endothelial cells (VECs) and they play a wide variety of critical roles in the control of vascular function. VECs are mostly susceptible to changes in blood composition and in blood flow as the interface between blood and tissue.1 Endothelial cell injury (EC injury) is integrally associated with inflammatory events which culminate in vascular damage. The damage to ECs appears to be an initial event which is characterized by altered shape, increased permeability, edema, intimal hyperplasia, necrosis and occlusion. Causes of EC injury may include shear stress, pharmacological reagents, immune complexes, circulating antibody deposition, and so on.2 Thrombomodulin (TM), a transmembranous glycoprotein acting as a receptor for thrombin,3 plays an important role as an anticoagulant protein on the blood vessel wall.4 Expression of TM on the surface of VECs is tightly regulated to maintain homeostasis and to ensure a rapid and localized hemostatic and inflammatory response to injury.5 Soluble thrombomodulin (sTM) is found after EC injury as shown by several in vitro studies. It is thought to be a cleaved form of tissue TM with loss of parts of the transmembrane domain and the cytoplasmatic tail,6 although its structure is not fully clear. TM has been regarded as a reliable marker of EC injury in vitro.7,8 Study on EC injury including evaluation of relevant markers can provide important information for early diagnosis and therapy of vasculopathy such as phlebophlogosis, atherosclerosis and thrombus. It is also valuable to cancer chemotherapy and other disease treatment in which some


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therapeutic drugs causing EC injury are usually used. The classical techniques for evaluation of sTM level involve

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Cr release method,9 enzyme-linked immunosorbent assay (ELISA),10 gel

electrophoresis followed by immunoblot analysis11 and immunoaffinity chromatography.12 Some special labels (e.g., radioactive isotope and fluorescent dye), expensive instruments and skilled operators are usually required in these measurements. Quartz crystal microbalance (QCM) has been widely used as a standard non-invasive tool to measure some real-time chemical reactions and bio-molecular interactions due to its satisfactory performance, such as, low cost, label-free, high sensitivity, rapid and facile operation, etc. In the liquid phase the QCM response is sensitive not only to mass loading but also to changes in solution density and viscosity near the electrode. The measured biological objects using QCM analysis include proteins, enzymes, antibody/antigen, nucleic acids, cells, and bacteria.13,14 Shons et al. first reported the QCM detection of cow serum IgG antibody in 1971,15 which initiates the development of the QCM immunoassay.16,17 However, the QCM immunosensors has been challenged because the direct QCM assay is not suitable for the determination of small molecules or analytes with extremely low detection limit. Hence, some strategies have been developed to improve the detection sensitivity of QCM immunosensors. Ultrasensitive QCM measurements in the liquid phase could be achieved by using high-frequency crystal,18,19 increasing the effective area of the electrode surface with porous films, molecular imprinted polymers, multilayers, etc,20,21 and realizing the mass-amplification on the electrode surface via the introduction of nanoplatforms such as nanoparticles22-25 and vesicles.26,27 To our knowledge, to date there are no reports on application of QCM for the determination of TM and the investigation on the relationship of sTM with EC injury. TM with extremely low concentration may not result in the obvious QCM signals due to the limit of the method


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sensitivity although the direct capture of TM molecules on the TM antibody modified QCM gold electrode (Ab/Au) can be achieved. So an indirect method of the TM detection was presented using nanomaterials in the present work. Anti-antibody (AAb) and bovine serum albumin (BSA) were bound to gold nanoparticles (GNPs) to construct BSA-GNPs-AAb nanocomposites and the real-time binding process of the prepared nanocomposites on Ab/Au was monitored using the QCM technique. TM molecules were beforehand introduced on the Ab/Au surface and their negative effect on the following capture of the nanocomposites was investigated. Label-free and sensitive detection of TM and the analysis on the release of sTM by injured VECs were performed using QCM measurement based on the immunoreaction. The mass-amplification on the QCM electrode surface was realized by using BSA-GNPs-AAb nanocomposites and the QCM signals were effectively improved. EXPERIMENTAL SECTION Materials and apparatus. Thrombomodulin, rabbit anti-human TM mAb (Ab) and goat antirabbit immunoglobulin (H+L) (anti-antibody, AAb) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (China). L-cysteine (Cys, Sigma-Aldrich) and bovine serum albumin (BSA, Sigma-Aldrich) was used as received. HAuCl4 and trisodium citrate were purchased from Shanghai Chemical Reagent Co., Ltd (China). Phosphate buffer solution (PBS, pH 7.4) consisting of 136.8 mmol L-1 NaCl, 9.7 mmol L-1 Na2HPO4, 2.7 mmol L-1 KCl and 1.5 mmol L-1 KH2PO4 was used in the experiments. Other chemicals were of analytical reagent grade and all aqueous solutions were prepared in Milli-Q ultrapure water. Resonant frequencies (f0) were acquired real time by a research QCM (Maxtek Inc., USA). The QCM sensor consists a thin AT-cut quartz crystal wafer with one gold electrode on each side (9 MHz, non-polished, 6-mm diameter). It was mounted on the side of a self-prepared


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polypropylene measuring chamber and immobilized between two biocompatible silicon O-rings to allow only one side of the electrode to be exposed to the liquid. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments were performed with a CHI660C electrochemical workstation (CH Instruments, China) by using a three-electrode electrolytic cell. The QCM gold electrode was used as the working electrode (WE). A saturated KCl calomel electrode (SCE) served as the reference electrode (RE) and all potentials in this work were cited with respect to it. A platinum plate served as the counter electrode (CE). The absorption spectra were recorded on a UV-2450 UV–vis spectrophotometer (Shimadzu, Japan). A Nicolet Nexus 670 FTIR spectrometer (Nicolet, USA) was employed for the infrared spectral analysis. The sizes of gold nanoparticles (GNPs) and BSA-GNPs-AAb nanocomposites were characterized ex situ by a Tecnai G2 20ST transmission electron microscope (TEM, FEI, USA). The images of the modified QCM gold electrode surface were obtained with an S-4800 scanning electron microscope (SEM, Hitachi, Japan). The cell-modality observation was performed with an inverted optical microscope (OLYMPUS CKX41, Japan). Preparation of BSA-GNPs-AAb nanocomposites. Colloidal GNPs were prepared according to the literature.28 Briefly, a 50 mL aqueous solution of 0.01% HAuCl4 (w/w) was heated to boiling in a round-bottom flask. 2 mL of 1% trisodium citrate was added to this solution drop by drop under vigorous stirring. The color of the solution changed from grey, blue, purple, to wine red, indicating the formation of gold nanoparticles. Then, the mixed solution was stirred continuously until it was cooled to room temperature and the prepared gold nanoparticles were stored in a brown glass bottle at 4 oC for further use. The preparation of BSA-GNPs-AAb nanocomposite suspension was performed as following: while gently stirring, 200 μL of AAb (100 μg mL-1) was added into 2 mL GNPs suspension


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(adjusted to pH 7.4 with 0.1 M NaOH) and continuously stirred for at least 5 h. The mixed solution was centrifugated at 13000 rpm for 20 min and the precipitate was dispersed with 1 mL pH 7.4 PBS. Then 200 μL of 5% BSA solution was added to cover the nonspecific sites. After stirring for at least 5 h, the solution was centrifugated at 13,000 rpm for 20 min. The purplish red precipitate was re-dispersed in 1 mL pH 7.4 PBS. Anti-antibodies and BSA were bound to gold nanoparticles based on Au-S bonding rather than physical adsorption of these biomolecules. The similar antibody-gold nanoparticle composites prepared by this method have been employed in many reports. 23,29-31 Randomness might be involved in the covalent bonding of biomolecules on GNPs in the preparation of the nanocomposites, which was hard to control. Surely it was difficult to ensure that the prepared BSA-GNPs-AAb nanocomposites in different batches were completely identical. But the prepared BSA-GNPs-AAb nanocomposites in same batch owned the satisfactory uniformity and could it be stored at 4 oC for more than 1 month without significant decrease in activity. Preparation of the chemically modified electrode and measurement procedure. To remove possible contamination, the QCM gold electrode was cleaned with Piranha solution (30% H2O2: H2SO4 3:7). Then the surface was thoroughly rinsed with ultrapure water and blown dry with a stream of nitrogen gas. The assembly process on the electrode surface and the measurement principle are shown in Scheme 1. The freshly cleaned QCM gold electrode was immersed into 5 mM Cys solution for overnight. Subsequently, the Cys-modified QCM electrode was immersed in 12% glutaraldehyde (GA) aqueous solution for 0.5 hour. Next, 5 μL of 100 μg mL-1 Ab solution was added on the dry GA/Cys-modified gold surface and cultured for 2 hours. Finally, 5% BSA was employed to block the non-specific sites. The prepared electrode was


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defined as Ab/Au. Before the QCM measurement, the TM solution with different concentration was casted on the Ab/Au surface and the modified electrode was marked as Ab-TM/Au. The electrode was gently washed with ultrapure water and then nitrogen-dried after each assembly process. The Ab/Au or Ab-TM/Au was mounted on the side of the measuring chamber and then 1.5 mL of pH 7.4 PBS was added. The QCM signals were obtained under stirring condition. As soon as the QCM readout became steady, 100 μL of BSA-GNPs-AAb nanocomposite suspension was added evenly. The resonant frequency shift (Δf0) derived from the capture of the nanocomposites was monitored up to 6 hours. If TM were beforehand casted on the Ab/Au surface, the steric hindrance would play a negative role in the binding of BSA-GNPs-AAb nanocomposites with antibodies immobilized on the QCM gold electrode surface, resulting in the decline of the Δf0 response. So it is feasible to develop a label-free method for the quantitative

Δf0 /Hz

TM detection.

t /min BSA-GNPs-AAb/Ab/Au

1 Glutaraldehyde

Cysteine

2

Au

3

Δf0 /Hz

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

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Ab/Au

t /min Ab-TM/Au BSA-GNPs-AAb/Ab-TM/Au

GNPs

BSA

TM

Rabbit anti-human TM mAb (Ab)

Goat anti-rabbit IgG (AAb)

Scheme 1. The assembly process of the sandwich structure on QCM gold electrode surface and the measurement principle.


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Cell culture and analysis. Human umbilical vein endothelial cell line, HUVEC-C cells were obtained from Xiangya School of Medicine, Central South University, China and they were incubated using DMEM/Low Glucose growth medium (HyClone) supplemented with 10% fetal bovine serum (FBS), in an incubator (5% CO2, 37 oC). HUVEC-C cells were seeded in 96-well plate at the concentration 5×104 cells mL-1. After 12 hours when the cells spread over the substrate, 10 μL of H2O2 was added in the wells and the cells were continuously cultured for 24 hours. 5 μL of growth medium was removed using a micropipettor at different time and casted on Ab/Au surface. The modified QCM gold electrode was subsequently employed to the next QCM measurement for capturing BSA-GNPs-AAb nanocomposites. Regeneration of the electrode. The used QCM gold electrode surface was cleaned with Piranha solution to remove the protein and nanocomposite coating. After rinsed thoroughly with ultrapure water, the electrode was subjected to a voltammetric treatment in 0.2 M HClO4 aqueous solution until cyclic voltammograms were reproducible. Then the surface was thoroughly rinsed and blown dry with a stream of nitrogen gas. After cleaned in this way, the QCM gold electrode could be regenerated and used repeatedly with good recovery of its initial f0 value. RESULTS AND DISCUSSION Characterization of BSA-GNPs-AAb nanocomposites. Figure 1A shows the photos of GNP and BSA-GNPs–AAb nanocomposite suspension. Wine red colloidal gold solution was observed and the nanocomposite solution presented purplish red. It means that the size of GNPs became larger after capturing biomacromolecules. Figure 1B and 1C show the TEM images of GNPs and BSA-GNPs-AAb nanocomposites, respectively. GNPs with the average diameter of 18 nm were linked each other. By contrast, BSA-GNPs-AAb nanocomposites were highly monodispersed


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and their size was relatively increased. It suggests that biomacromolecules were successfully assembled on the surface of GNPs and their electronegativity resulted in the electrostatic repulsion between the nanocomposites.

Figure 1. (A) Photos of GNPs (left) and BSA-GNPs–AAb nanocomposite suspension (right). TEM images of GNPs (B) and BSA-GNPs–AAb nanocomposites (C). Figure 2A shows the UV-vis spectra of GNPs, BSA, AAb and BSA-GNPs-AAb nanocomposites in aqueous solution from 200 to 800 nm, respectively. GNPs had a characteristic absorbance peak at about 523 nm. A prominent absorption peak at 273 nm, which is attributed to Tyr and Trp residues,32 can be found on the spectrum of BSA. A similar absorption peak at 277 nm was presented on the spectrum of AAb. The spectrum of BSA-GNPs-AAb nanocomposites appeared to be an overlapping of two spectra for the GNPs and AAb accompanied with red shift of the absorption peak in the visible region. FTIR spectra of GNPs, BSA, AAb and BSA-GNPs– AAb nanocomposites in the range of 4000-400 cm-1 are shown in Figure 2B. The characteristic peaks of GNPs at ~1396 and ~1630 cm-1 were ascribed to the symmetric and asymmetric stretching vibration of –COO-, respectively, which means that citrate was adsorbed on GNPs that were prepared based on the redox reaction using HAuCl4 and trisodium citrate. AAb and BSA had almost same bands. Two bands at ~1647 and ~1532 were attributed to the absorption bands I and II of amino acids, respectively. The absorption band at ~1166 cm-1 was derived from the C– N stretching vibration. The absorption bands at ~1647, ~1532, ~1396 and ~1166 cm-1 can be all


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found in the spectrum of BSA-GNPs-AAb nanocomposites. The above UV-vis and FTIR spectra measurement results all indicate that the prepared nanocomposites were composed of GNPs, AAb and BSA. 0.5

A

Absorbance

0.4 0.3 0.2 0.1

d

b a

c 0.0 200

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Wavelenth /nm

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1166

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1647 1647 1532 1532 1396

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a

Transmittance / %

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B

1000 -1

Wavenumber /cm

Figure 2. UV-vis absorption spectra (A) and FTIR spectra (B) of GNPs (a, red line), BSA (b, green line), AAb (c, pink line) and BSA-GNPs-AAb nanocomposites (d, blue line). Negative effect of TM on capture of BSA-GNPs-AAb nanocomposites on the TM antibody-modified electrode. Figure 3A, 3B and 3C show the SEM images of Ab/Au, BSAGNPs-AAb/Ab/Au and BSA-GNPs-AAb/Ab-TM/Au surface, respectively. A rough and thick protein coating can be observed on Ab/Au (shown in Panel A). One can find from Panel B that not a few nanoparticles were distributed uniformly on the Ab coating-modified electrode surface. It indicates the capture of BSA-GNPs-AAb nanocomposites based on the immunoreaction of


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AAb with antibody. Furthermore, the nanocomposite amount on Ab-TM/Au (shown in Panel C) was much less than that on Ab/Au (shown in Panel B). It suggests that the binding of the nanocomposites was effectively inhibited in the presence of the TM molecules.

Figure 3. SEM images of Ab/Au surface (A), BSA-GNPs-AAb/Ab/Au surface (B) and BSAGNPs-AAb/Ab-TM/Au surface (C). Magnification: 50000×. Figure 4A and 4B exhibit the cyclic voltammograms and electrochemical impedance spectroscopy of QCM gold electrode before and after Ab-Ag immunoreaction in pH 7.0 PBS using ferri-/ferrocyanide probe, respectively. A couple of reversible redox peaks and a small high-frequency semicircle diameter in the Nyquist plot, which presents the charge transfer resistance (Rct), can be found on the bare QCM gold electrode. The assembly of Ab resulted in the decrease of the peak currents and increase of the Rct value, respectively, indicating that the antibodies were immobilized on modified electrode. The above two electrochemical parameters were changed to a greater extent with the binding of TM. It means that the bound antigen molecules resulted in the strong steric hindrance effect on the electrode surface, which efficiently blocked the charge transfer of ferri-/ferrocyanide probe. Likewise, this steric hindrance effect could lead to the significant inhibition for the AAb-Ab reaction and the capture of BSA-GNPsAAb nanocomposites (as shown in Figure 3C).


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20

a b

I /μA

A

c

10

0

-10

-20 -.1

0.0

.1

.2

.3

.4

.5

E /V vs SCE 1000

B

800

-Z'' /Ω

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


600 c b

400 a

200 0 0

500

1000

1500

Z' /Ω

Figure 4. Cyclic voltammograms (A) and Electrochemical impedance spectra (B) of Au (a), Ab/Au (b) and Ab-TM/Au (c) in pH 7.0 PBS containing 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6, and 0.1 M Na2SO4. The scan rate: 50 mV s-1. AC frequency range 100 kHz−1 mHz, amplitude 5 mV, DC bias 0.20 V vs. SCE QCM detection of TM. The negative effect of TM on the capture of BSA-GNPs-AAb nanocomposites was employed to detect the TM concentration in this work. Some factors, which affecting the assembly of the nanocomposites on the QCM gold electrode surface, such as the dilution times of BSA-GNPs-AAb nanocomposites and the incubation time of TM, were investigated, respectively, and the results are presented in Figure 5. Here the frequency in air of Ab-TM/Au and that of BSA-GNPs-AAb/Ab-TM/Au were represented as f0(air,1) and f0(air,2), respectively, and f0(air,1)>f0(air,2). Figure 5A shows the effect of the dilution times of BSA-GNPs-


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AAb nanocomposites on the frequency-change in air (Δf0(air)), which is the difference between f0(air,1) and f0(air,2). One can find that the Δf0(air) value was slightly increased when the nanocomposites suffered two-fold dilution and then it was quickly declined at the higher dilution times. It suggests that BSA-GNPs-AAb nanocomposites with high concentration might lead to the steric hindrance effect on Ab-TM/Au surface, which went against the effective capture of these nanocomposites. On the other hand, the effective loading that could dominate the QCM response change depended on the adequate amount of BSA-GNPs-AAb nanocomposites on the electrode surface. Thus, the optimal dilution times was selected at 2 in order to obtain the maximum Δf0(air) value. Figure 5B shows a series of the Δf0(air) values due to the capture of BSAGNPs-AAb nanocomposites with the same concentration on Ab-TM/Au that was casted beforehand by TM for different time. The Δf0(air) value was decreased with the increasing incubation time of TM from 10 to 120 min and then suffered a little enhancement when the incubation time was further prolonged. Enough incubation time was beneficial to the stable immobilization of TM. As a result, the capture of the nanocomposites would be inhibited and the Δf0(air) value was declined. The optimal condition for the incubation time of TM was chosen at 120 min since this work focused on the TM detection based on the inhibition effect of TM on the capture of BSA-GNPs-AAb nanocomposites.


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500

A

Δf0(air) /Hz

400 300 200 100 0 0

10

20

30

40

Diluted times of BSA-GNPs-AAb 800

B 700

Δf0(air) /Hz

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


600

500

400 0

60

120

180

Incubation time of TM proteins /min

Figure 5. Effects of the dilution times of BSA-GNPs-AAb nanocomposites (A) and the incubation time of TM proteins (B) on the frequency-change in air of Ab-TM/Au before and after immobilization of BSA-GNPs-AAb nanocomposites (Δf0(air)). Results are presented as mean ± SD (error bar) of triplicate experiments. The real-time frequency monitoring on the capture of BSA-GNPs-AAb nanocomposites on Ab/Au modified with TM at different concentration, Ab-TM/Au, was performed and the results are shown in Figure 6A. Before the cell addition, the QCM was initially equilibrated with pH 7.4 PBS for 40 minutes, until stable baseline for Δf0 was achieved. It is found that the Δf0 value was decreased slowly with the addition of the nanocomposites and it kept declining during the whole measurement process. The decline rate of the frequency was far smaller than that induced by adsorption of precipitate33 and protein34 as the latter was a random process on the QCM electrode


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surface. Moreover, the changing extent of f0 on Ab/Au (curve a) was larger than that on AbTM/Au (curves b-g) within the same time and the latter was gradually decreased with the enhancement of the TM concentration. It means that the beforehand immobilized TM molecules exerted negative effect on the capture of the nanocomposites though the binding sites for antigens were different with that for anti-antibodies on TM antibodies. The above conclusion is in complete agreement with the SEM measurement results in Figure 3. The more TM molecules were on the electrode surface, the less binding spaces for the nanocomposites were remained. The relative frequency-shift, RFS, is defined as, RFS =

Δf 0(blank) − Δf 0(TM) Δf 0(blank)

(1)

where Δf0(blank) and Δf0(TM) are the QCM-frequency shifts at the sixth hour after addition of BSAGNPs-AAb nanocomposites on Ab/Au and Ab-TM/Au, respectively. As shown in Figure 6B, the RSF value exhibited a linear response with respect to lgcTM over the TM concentration from 10 to 5000 ng mL-1. The regression equation was RFS = 0.2155lgcTM + 0.0036 with a high correlation coefficient of 0.977 and the detection limit was found to be 2 ng mL-1 estimated at a signal-to-noise ratio of 3. Hence, a sensitive and label-free sensor for the quantitative detection of TM could be developed using piezoelectric technique.


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60

BSA-GNPs-AAb

0 g

Δf0 /Hz

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-120 -180 -240 -300

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B

.6 .4 .2 0.0 .5

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lgcTM /lg(ng mL )

Figure 6. (A) The real-time Δf0 responses of Ab/Au modified with TM at the concentration of 0 (a), 10 (b), 50 (c), 100 (d), 500 (e), 1000 (f) and 5000 (g) ng mL-1 (Ab-TM/Au) to the addition of BSA-GNPs-AAb nanocomposites. (B) Plot of relative frequency-shift (RFS) vs lgcTM. Results are presented as mean ± SD (error bar) of triplicate experiments. Study on sTM release by injured endothelial cells. In this work, H2O2 was selected as a cause model to study the relationship between EC injury and sTM release. When HUVEC-C cells were in their growth phases, H2O2 was added into the growth medium and its final concentration was 0.75 mM. HUVEC-C cells in growth medium in the absence and presence of H2O2 were observed at the different time after the addition of H2O2 and their microscopic photos are shown in Figure 7A-D, respectively. The normal HUVEC-C cells were polygon- or spindleshaped and basically distributed on the substrate as a monolayer. One can find the obvious


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transformation of the cellular shapes after stimulation by H2O2. Contraction occurred among a small population of HUVEC-C cells that became shiny spheroids, and most of the cells gradually became flat circle-like structure with vague cellular outline. This changing trend was more and more obvious with the increase of contact time of the cells with H2O2. Fourteen hours later, no living cells were observed and only a large number of vesicles were presented on the substrate. The above results indicate that H2O2 effectively resulted in injury of these vascular endothelial cells. It is reported that H2O2 can decompose into hydroxyl radicals that readily react with and damage vital cellular components via oxidative stress.35 Cell injury through base modifications and strand breakage in genomic DNA36 as well as apoptosis induction37 are closely related to accumulation of H2O2.

Figure 7. Microscope images (200×) of HUVEC-C cells incubated in growth medium at different time after addition of H2O2. (A) 0 mM H2O2, 14 h. (B) 0.75 mM H2O2, 2 h. (C) 0.75 mM H2O2, 8 h. (D) 0.75 mM H2O2, 14 h. Some co-existing components in DMEM growth medium supplemented with 10% FBS, such as glucose, folic acid and BSA, were analyzed, respectively, to investigate possible interferences from cell culture condition. 5 μL of pH 7.4 PBS, 500 ng mL-1 TM, 5 mM glucose, 10 μg mL-1 folic acid and 100 μg mL-1 BSA were introduced beforehand on several Ab/Au electrodes,


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respectively. The real-time frequency shifts of these modified QCM electrodes to the capture of BSA-GNPs-AAb nanocomposites were measured and the results are shown in Figure 8A. One can find that only the Ab/Au beforehand treated by TM resulted in a relatively smaller -Δf0 value at the sixth hour after addition of BSA-GNPs-AAb nanocomposites and the changing trend of the Δf0 curves on other electrodes were almost identical. It demonstrates that glucose, folic acid and BSA had no effect on the subsequent immobilization of the nanocomposites on Ab/Au because these analytes could not be bound to Ab/Au. Hence, the developed QCM immunosensor could present a high specificity for the TM detection in the presence of these co-existing components. Growth media in cell incubation wells shown in Figure 7A-D were introduced on four Ab/Au electrodes, respectively, and the modified electrodes, which were defined as Ab-sTMA/Au, AbsTMB/Au, Ab-sTMC/Au, and Ab-sTMD/Au, were employed to capture BSA-GNPs-AAb nanocomposites, respectively. The real-time QCM monitoring results are shown in Figure 8B. One can find from curve a that the -Δf0 value on Ab-sTMA/Au at the sixth hour after addition of BSA-GNPs-AAb nanocomposites was about 200 Hz, smaller than that on Ab/Au at the same time, ~260 Hz (shown by curve a in Figure 6A). It suggests that a small quantity of soluble TM molecules, which might be derived from the normal metabolism of HUVEC-C cells, were presented in growth medium and they could be immobilized on the antibody modified QCM gold electrode, inhibiting the next binding of the nanocomposites. It is interesting to be found that the -Δf0 responses due to the capture of BSA-GNPs-AAb nanocomposites on Ab-sTMB/Au, AbsTMC/Au, and Ab-sTMD/Au were much smaller than that on Ab-sTMA/Au and the three formers were decreased in sequence. Several previous clinical studies have shown that plasma levels of sTM are increased in various diseases associated with endothelial cell injury or proteolytic activity on the endothelial cell surface.38-40 The above QCM experimental results proved the


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gradual release of sTM from the injured HUVEC-C cells under the durative stimulation of H2O2 and the successive enhancement of the bound sTM molecules on Ab-sTMB/Au, Ab-sTMC/Au, and Ab-sTMD/Au. As a result, it was more and more difficult for BSA-GNPs-AAb nanocomposites to be captured on these electrode surfaces. 60

BSA-GNPs-AAb

A

0

b

-120 -180

a

c d e

-240

240 160

b

-300

320

80

−Δf0 /Hz

Δf0 /Hz

-60

c/d/e a

0

-360

Ab/Au 0

100

200

300

400

t /min BSA-GNPs-AAb

B

0

Δf0 /Hz

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

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d -80 c b -160 a -240 0

100

200

300

400

t /min

Figure 8. (A) The real-time Δf0 responses of Ab/Au treated with reagent blank (a), 500 ng mL-1 TM (b), 5 mM glucose (c), 10 μg mL-1 folic acid (d) and 100 μg mL-1 BSA (e) to the addition of BSA-GNPs-AAb nanocomposites. Insert: the -Δf0 values at the sixth hour after addition of BSAGNPs-AAb nanocomposites on the above Ab/Au electrodes. (B) The real-time Δf0 responses to the addition of BSA-GNPs-AAb nanocomposites on Ab-sTMA/Au (a), Ab-sTMB/Au (b), AbB

sTMC/Au (c) and Ab-sTMD/Au (d). CONCLUSIONS


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The capture of the nanocomposites consisted of GNPs, anti-antibody and BSA on the TM antibody-modified QCM gold electrode could lead to the decrease of the resonant frequency in the real-time QCM monitoring. This capture was effectively inhibited by the strong steric hindrance effect derived from the immunoreaction between TM and the antibodies, which resulted in the reduction of the frequency changing extent. The relative frequency-shift was found to be closely related to the concentration of TM and the detection limit was down to ng mL-1 level. The fabricated QCM sensor was successfully employed to investigate the release of sTM by the injured endothelial cells. The developed label-free and sensitive QCM method based immunoreaction might provide a promising tool for monitoring the gene/protein release or expression occurring in some cellular processes. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Scientific Research Fund of Hunan Provincial Education Department (14A095) and the Open Sustentation Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (2014007).


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Δf0 /Hz

For Table of Contents Only

GNPs

t /min BSA

BSA-GNPs-AAb/Ab/Au

TM

Ab

Ab/Au

Δf0 /Hz

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


t /min

AAb Ab-TM/Au BSA-GNPs-AAb/Ab-TM/Au


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Label-Free and Sensitive Detection of Thrombomodulin, a Marker of

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