Quartz crystal microbalance immunoassay with dendritic amplification using colloidal gold immunocomplex

Sensors and Actuators B 114 (2006) 696–704

Quartz crystal microbalance immunoassay with dendritic amplification using colloidal gold immunocomplex Xia Chu a,b,∗ , Zi-Long Zhao a , Guo-Li Shen b,∗ , Ru-Qin Yu b a

Chemistry and Chemical Engineering College, Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), Hunan Normal University, Changsha 410081, PR China b State Key Laboratory for Chemo, Biosensing and Chemometrics, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, PR China Received 15 February 2005; received in revised form 10 May 2005; accepted 16 June 2005 Available online 2 August 2005

Abstract A novel dendritic amplification procedure has been developed for microgravimetric quartz crystal microbalance (QCM) immunosensing by the application of antibody-functionalized Au nanoparticles as the primary amplifying probe and a dendritic-type immunocomplex of protein A- and antibody-modified Au nanoparticles as the secondary amplifying probe. Goat anti-human immunoglobulin G (IgG) antibody is orientedly immobilized on the Au electrode surface of QCM through pre-assembled protein A and acts as the sensing interface to recognize the analyte, human IgG. The primary amplification of the recognition process is implemented via the interaction of the sensing interface with the antibody-functionalized Au nanoparticles, and the secondary dendritic amplification is performed through interaction with the immunocomplex of protein A- and antibody-modified Au nanoparticles. The dendritic-type amplified sensing procedure is also confirmed by the UV–vis absorption measurements. The frequency decreases of the primary amplified and the secondary amplified sensing process are observed to be linearly dependent upon the IgG concentration in the range of 10.9 ng ml−1 to 10.9 ␮g ml−1 with a detection limit of 3.5 ng ml−1 , while in the absence of the amplification processes, the antigen–antibody recognition event can only be detected for a IgG concentration as high as 10.9 ␮g ml−1 . © 2005 Elsevier B.V. All rights reserved. Keywords: Quartz crystal microbalance; Dendritic amplification; Immunoglobulin G; Protein-functionalized Au nanoparticle

1. Introduction In recent decades, quartz crystal microbalance (QCM)based piezoelectric immunosensors have found widespread applications in the analysis of clinical targets [1,2], the monitoring of environmental contaminants, such as pathogen and bacteria [3,4] and the detection of biomolecular interaction [5,6] due to its attractive performance, such as high specificity, low cost, ease of use and rapidness of detection. Despite of the success, the proliferation of immunosensors-based on QCM transduction for trace biological target detection is still challenged by its relatively low intrinsic ∗

Corresponding authors. Tel.: +86 731 8821916; fax: +86 731 8821916. E-mail addresses: [email protected] (X. Chu), [email protected] (G.-L. Shen). 0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.06.014

sensitivity. Extensive efforts have been made to further improve the sensitivity of the QCM-based immunosensors [6]. As far as the chemical approaches are concerned, two strategies are commonly utilized for the sensitivity enhancement in piezoelectric immunosensors. One is the so-called surface enhancement technique, which involves the increase of the effective area of the electrode surface or the improvement of the loading of active antibody or antigen molecules at the electrode surface via a suitable immobilization method. The other is the mass amplification technique, which comprises the deposition of an amplified mass on the electrode surface via a chemical route activated by the adsorbed analytical targets. The immobilization of antibodies and antigens not only produces a functionalized sensing interface but also determines the sensitivity as well as the regeneration ability

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of the QCM-based immunosensors. A most commonly used immobilization approach is performed through the covalent attachment of antibodies or antigens to a intermediate derivatizable substrate layer on the electrode surface, such as self-assembled monolayers [4,7], Langmuir–Blodgett (LB) films [8], polymeric coatings [9–11], electropolymerised films [12] and plasma polymerized films [13,14]. Immobilization was also implemented using the direct adsorption of antibodies or antigens on the electrode surface or the intermediate substrate layer, including specially thiolated antibodies or antigens [15], layer-by-layer assembled polyelectrolyte films [16], and assembled nanoparticle layer [17]. The immobilization methods that lead to sensitivity enhancement are generally characterized by an increased electrode surface and thus a substantially improved loading of antibodies or antigens. However, criticism toward these approaches lies in the fact that the immobilized antibodies or antigens are randomly oriented, implying that a part of immobilized antibodies or antigens are not accessible to the analytical targets. A strategy for oriented immobilization of antibodies or antigens then promises an ideal sensitivity enhancement due to the optimized presentation of the immobilized molecules that produces unobstructed recognition sites for the analytes. Oriented immobilization was achieved through the use of particular functional groups in the antibody Fc domain to orientedly link the antibody on the functionalized electrode surface [18,19]. These procedures generally comprised complicated and time-consuming operations for the pretreatment of the functional groups in the antibody Fc domain. Another approach to oriented immobilization of antibodies that was easy to operate and cost-efficient was to utilize the immobilized protein A that binds orientedly the antibody Fc domain with high affinity and selectivity [20–23]. The second sensitivity enhancement strategy, i.e. mass amplification, is generally implemented using the labeldependent assay format such that the mass of the analyte adsorbed on the electrode surface can be amplified though some chemical routes. Typical routes involve the utilization of a catalytic label that induces insoluble precipitates to deposit on the electrode surface [24] or the application of a massy label that have mass much larger than the analyte [25–27]. It was shown that a sandwich immunoassay using horseradish peroxidase (HRP) functionalized liposomes as the catalytic label resulted in a substantially improved sensitivity [24]. Also reported was a QCM transduction of the oligonucleotide-DNA sensing processes amplified using antibodies as the massy label [25]. Significant mass amplification procedure was described in a sandwich DNA detection using oligonucleotide-functionalized liposomes as the massy label [26]. Further mass amplification was achieved by use of the dendritic-type complex of biotinylated liposome and avidin as well as oligonucleotide-functionalized Aunanoparticle and DNA complex [26,27]. Implementation of QCM immunosensor for quantitative detection of antigenprotected nanoclusters was also described quite recently [23].

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In the present study, a novel dendritic mass amplification method for QCM immunosensing was developed by use of the antibody-functionalized Au nanoparticles for the primary amplification and the dendritic immunocomplex of protein A- and antibody-modified Au nanoparticles for the secondary amplification. Though the application of antigennanoclusters and antibody-nanoclusters to amplified QCM transduction of immunoreaction events seems very straightforward, to our knowledge no dendritic amplification procedure has been addressed in the QCM-based immunosensing field. To further optimize the immunosensing performance, the protein A-based oriented immobilization of antibodies was employed. Results obtained revealed that the sensitivity was substantially improved via the amplification step and a detection limit as low as 3.5 ng ml−1 was approached for a model analyte of human immunoglobulin G (IgG).

2. Experimental 2.1. Apparatus The piezoelectric quartz crystals (AT-cut, 8 MHz, gold electrode) were purchased from Shanghai Chenhua Equipment (Shanghai, China). To stabilize the oscillation frequency in solutions, one side of the crystals was sealed with O-ring of silicone rubber covered by a plastic plate to form an air compartment. The crystal was powered through an oscillator circuit constructed from a transistor–transistor logic integrated circuit (TTL-IC). The oscillation frequency was monitored with a high frequency counter (Model FC 1250, Wellstar). A magnetic stirrer (Model JB-2, Shanghai Analytical instruments, Shanghai, China) was used to gently agitate the solution in a laboratory-made reaction cell where the one-side-sealed crystal was mounted. A Model CSS 501 thermostat from Chongqing Experiment Equipments (Chongqing, China) was used to control the temperature. The UV–vis absorption spectra were measured using a Lamda 800 UV–vis spectrometer (Pekin-Elmer). Transmission electron microscopy (TEM, HITACHI-H800) was obtained from Hitachi Ltd. (Tokyo, Japan) and copper grids (no. 50–230) were purchased from the Scientific Instruments Ltd. of Chinese Academy of Sciences (Beijing, China). 2.2. Materials Human immunoglobulin G (human IgG), goat antihuman IgG antibody (specific to the ␥-chain of human IgG) and bovine serum albumin (BSA) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China). Protein A was purchased in the lyophilized form from China Biotechnology Co., Shanghai Institute of Biological Products (Shanghai, China). 3-(Aminopropyl)triethoxylsilane (APTES) was purchased from Sigma Chemicals Co. (USA). Chloroauric acid (HAuCl4 ), trisodium citrate and glutaraldehyde were

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purchased from Shanghai Chemical Reagents (Shanghai, China). All other reagents were of analytical reagent grade. Double-distilled water was used throughout the experiments. 2.3. Buffers and solutions The following buffers were used in this study: (a) phosphate buffer solution (PBS; 10 mM KH2 PO4 , 40 mM Na2 HPO4 , pH 7.38); (b) Tris-buffered saline (TBS; 20 mM Tris and 150 mM NaCl, adjusting pH to 8.0 with concentrated HCl); (c) TBS containing 0.1% BSA (TBS-BSA). Human IgG standard solutions were diluted from a stock solution (10.9 mg ml−1 ) with PBS. Goat anti-human IgG solution (1 mg ml−1 ) was prepared by dilution of a stock solution (4.45 mg ml−1 ) with PBS. The colloidal gold-labeled goat anti-human IgG antibody and the colloidal gold-labeled protein A were prepared and diluted with TBS-BSA. 2.4. Preparation of colloidal gold Au particles were prepared using a method previously reported [28] with slight modification. Briefly, in a 500 ml round-bottom flask, 250 ml HAuCl4 (0.01%) was brought to a boil with vigorous stirring. To this solution was rapidly added 3.75 ml of 1% trisodium citrate. The solution turned deep blue within 20 s and the final color changed to wine-red 60 s later. Boiling was pursued for an additional 10 min. The colloidal gold was stored in dark bottles at 4 ◦ C and could be used for more than half of 1 year to label the antibody and protein A. The UV–vis spectrum showed that the resulting colloidal solution had an absorption maximum at 520 nm. Transmission electron microscopy (TEM) indicated a particle size of 14 ± 2.0 nm (100 particles sampled). 2.5. Preparation of the protein A–colloidal gold conjugate

resuspending in 1 ml of TBS-BSA and collected after a second centrifugation at 17,390 × g for another 10 min. Finally, the conjugate was resuspended in 100 ␮l of TBS with 0.1% BSA added to minimize nonspecific adsorption and increase the stability of the conjugate during the immunoassay. Conjugates can be stored at 4 ◦ C for more than 1 month without significant decrease in activity. 2.6. Preparation of the antibody–colloidal gold conjugate Goat anti-human IgG antibody–colloidal gold conjugate, i.e. the antibody-functionalized Au nanoparticles, was prepared as described previously [31] and briefly given as follows: while gently stirring, goat anti-human IgG antibody solution (containing 30 ␮g antibody) was added into 1 ml of colloidal gold solution (adjusted to pH 9.0 with 0.1 M NaOH). The following processes were the same as those described in the preparation of the protein A–colloidal gold conjugate. Finally, the sediment of the immunogold colloid was resuspended in 100 ␮l of TBS-BSA. Conjugates can be stored at 4 ◦ C for more than 1 month without significant decrease in activity. 2.7. Preparation of the colloidal gold immunocomplex The colloidal gold immunocomplex, i.e. the dendritictype immunocomplex of protein A- and antibodymodified Au nanoparticles, was prepared by mixing the antibody–colloidal gold conjugate and the protein A–colloidal gold conjugate in appropriate volume ratio. The mixture was incubated for 2 h at 37 ◦ C. After a centrifugation at 17,390 × g for 10 min, the supernatant was removed and the sediment of the colloidal gold immunocomplex was resuspended in 100 ␮l of TBS-BSA and stored at 4 ◦ C for use. 2.8. Measurement procedure

The protein A–colloidal gold conjugate, i.e. the protein A-functionalized Au nanoparticles, was prepared according to a documented method [29,30] with slight modification. Firstly, the amount of protein A that was necessary to coat the exterior of the gold particles was determined according to the observed dispersion stability in the flocculation test [29]. For 20 nm colloidal gold, the optimum amount of protein A for coating the gold nanoparticles was 30 ␮g/1 ml colloidal gold solution. In the preparation, the protein A solution (containing 30 ␮g protein A) was added to 1 ml colloidal gold solution (adjusted to pH 6.0 with 0.1 M NaOH) while gently agitating. The mixture was incubated at room temperature for 1 h. Then 200 ␮l of 5% BSA was added into the mixture to stabilize the colloidal gold solution and the mixture was incubated for an additional 30 min. Upon centrifugation at 17,390 × g for 10 min, two phases were obtained: a clear to pink supernatant of unbound protein A and a dark red, loosely packed sediment of conjugate. The supernatant was discarded and the soft sediment of conjugate was rinsed by

The one-side-sealed crystal was cleaned by pipetting 50 ␮l fresh piranha solution (one part 30% H2 O2 , three parts H2 SO4 ) on the gold electrode surface for 10 min followed by rinsing with double-distilled water and air-dried. The crystal’s frequency was monitored and recorded in the air at room temperature. The solution of protein A (20 ␮l, 1 mg ml−1 ) was applied on the cleaned surface of gold electrode and incubated at 4 ◦ C for over night. After removing the solution, the crystal was washed with 0.1 M NaCl and doubly distilled water two times each for 1 min and dried in air at room temperature. After the assembly of protein A on Au electrode surface, the goat anti-human IgG (20 ␮l, 1 mg ml−1 ), human IgG standard solution (20 ␮l), colloidal gold-labeled goat anti-human IgG antibody (20 ␮l) and the optimized colloidal gold immunocomplex (20 ␮l) were applied in turn on the resulting surface and allowed to react for 1 h at 37 ◦ C. After each adsorption, the crystal was washed and dried in the

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aforementioned manner and the crystal’s frequency was monitored and recorded in the air. In the liquid-phase continuous monitoring experiment, the protein A-functionalized QCM probe was inserted into the reaction cell with 3.0 ml PBS (pH 7.0, containing 0.9% NaCl). Under gentle stirring, goat anti-human IgG antibody (100 ␮l, 1 mg ml−1 ) was introduced into the reaction cell after the stabilization of resonance frequency (shift less than 1 Hz min−1 ) and the frequency changes of the crystal were recorded until equilibrium was reached. The crystal was washed with 0.1 M NaCl and doubly distilled water and then inserted into reaction cell. The frequency changes resulting from the addition of human IgG standard solution (100 ␮l), colloidal gold-labeled goat anti-human IgG antibody (100 ␮l) and the optimized colloidal gold immunocomplex (100 ␮l) were monitored and recorded as mentioned above. After each immunoassay, the used protein A-immobilized QCM interface was regenerated by rinsing in the glycine–HCl buffer (0.1 M, pH 2.3) and then washed in the ultrasonic water cleaner, each for 15 min. The regenerated protein Aimmobilized interface could accomplish up to seven repetitive assays without significant loss of detection sensitivity. 2.9. UV–vis absorption spectra measurement The goat anti-human IgG antibody was immobilized on a glass support as depicted in Scheme 1. The glass slide (1 cm × 3 cm) was cleaned thoroughly by dipping in 1.2 M NaOH for 30 min, washed with distilled water and further soaked in normal HCl for 2 min. After washing extensively with distilled water, the glass slide was then reacted with 5% APTES solution in pH 4.5 HAc–NaAc buffer in a plastic vessel at room temperature for over night. After incubation, the glass slide was washed with distilled water. The APTESmodified glass was then dipped in 2.5% glutaraldehyde water solution for 2 h at room temperature. The glass was washed with water and air-dried. Finally, 1 ml of 1:10 goat anti-human

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IgG antibody was applied on the glass slide surface and incubated at 37 ◦ C for 1 h. The glass was further rinsed in 0.5 M NaCl solution followed by washing with distilled water. After air drying, the UV–vis absorption spectrum of the glass slide was measured. The antibody-modified glass slide was then reacted with human IgG, colloidal gold-labeled antibody and colloidal gold immunocomplex, respectively, and the UV–vis absorption spectra were measured. 3. Results and discussion The principle of the QCM-based human IgG sensing with dendritic amplification is depicted in Scheme 2. Protein A is a cell wall protein that forms stable complexes with Au0 through van der Waal interactions with relatively high affinity. When protein A is firstly adsorbed on the Au-electrode surface of QCM, this results in a monolayer loading of protein A on the electrode surface. After gold surface is blocked by TBS containing 0.1% BSA, the goat anti-human IgG antibody solution is added to react with the protein A-coated surface. Because protein A binds specifically to the Fc domain of the antibody, a sensing interface is obtained with oriented immobilization of the antibodies such that the antigen-reactive sites are unobstructed and the protein Areactive sites are masked. Interaction of the sensing interface with the analyte, human IgG, leads to the antigen–antibody immunocomplex. The primary amplification of the sensing process is performed by the interaction of the surface with the antibody–colloidal gold conjugate, producing excessive antibody on the electrode surface as reactive sites for the secondary amplification. The secondary, dendritic-type amplification is implemented by the interaction of the resulting surface with the colloidal gold immunocomplex prepared beforehand, which comprises the dendritic complex of the antibody-functionalized gold nanoparticles and the protein A-functionalized gold nanoparticles. The binding of excessive antibody on the surface to the excessive protein A

Scheme 1. Immobilization of the goat anti-human IgG antibody on a glass support.

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Scheme 2. Dendritic amplification of the antigen–antibody recognition event using antibody-functionalized Au nanoparticles followed by the dendritic complex of protein-functionalized Au nanoparticles and antibody-functionalized Au nanoparticles.

in the dendritic complex then enables the secondary amplification. As a result, the frequency response associated with the antigen–antibody binding event is amplified by the mass accumulated in the primary sandwich immunoreaction and the secondary deposition of the dendritic immunocomplex on the electrode surface, offering a substantial amplification route for the sensing of the analytical targets. 3.1. Liquid monitoring experiment Fig. 1 shows the typical frequency changes of the quartz crystal functionalized with protein A as a result of a cascade of interactions with goat anti-human IgG antibody, human IgG, colloidal gold-labeled antibody and colloidal gold immunocomplex. A frequency decrease of about −76 Hz was observed after interaction with 33 ␮g ml−1 of goat antihuman IgG antibody, as is shown in Fig. 1A. Subsequent association of 10.9 ␮g ml−1 of human IgG with the sensing interface resulted in an additional frequency response of about −42 Hz, whereas the addition of 10.9 ␮g ml−1 of rabbit IgG led to a frequency decrease of only −5 Hz (Fig. 1B). These results indicated that all protein A active sites were saturated with goat anti-human IgG antibody. Treatment of the resulting interface with the primary amplification probe, i.e. antibody–colloidal gold conjugate, yielded a frequency decrease of about −245 Hz (Fig. 1C), indicating that the

binding of the antibody–colloidal gold conjugate offered a significant amplification for the sensing of IgG. Additional treatment of the interface with the secondary amplification probe, i.e. the colloidal gold immunocomplex, resulted in a second amplification and a very large frequency change of about −501 Hz was observed (Fig. 1D). One also observed from Fig. 1C and D that the treatment of the sensing interface with the primary and the secondary amplification probes merely gave frequency changes of only about −47 and −37 Hz, respectively, which were attributed to the nonspecific association of the protein to the interface. These results revealed that the introduced dendritic amplification procedure for immunosensing was high specific and sensitive. 3.2. Optical measurement of dendritic amplification The dendritic-type amplification for the sensing of human IgG was further confirmed by the UV–vis absorption measurements. The goat anti-human IgG antibody was immobilized on a glass support as shown in Scheme 1. Treatment of the antibody-immobilized glass with the analyte, human IgG, and subsequently with the antibody–colloidal gold conjugate, resulted in the spectrum shown in Fig. 2, curve b, which reveals a characteristic plasmon resonant absorption band of Au nanoparticles with a maximum absorbance about 0.02 (the blank absorbance was

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Fig. 1. Time-dependent frequency changes of the quartz crystal. (A) Addition of goat anti-human IgG antibody (final concentration, 33 ␮g ml−1 ) to reaction cell inserted with protein A-assembled quartz crystal; (B) addition of rabbit IgG (10.9 ␮g ml−1 ) (1) or human IgG (10.9 ␮g ml−1 ) (2) to reaction cell inserted with protein A/IgG antibody-assembled quartz crystal; (C) addition of colloidal gold-labeled antibody to reaction cell inserted with protein A/IgG antibodyassembled quartz crystal (1) or protein A/IgG antibody/IgG antigen-assembled quartz crystal (2); (D) addition of colloidal gold immunocomplex to reaction cell inserted with protein A/IgG antibody-assembled quartz crystal (1) or protein A/IgG antibody/IgG antigen/colloidal gold-labeled antibody-assembled quartz crystal (2). All measurements were performed in 3 ml of pH 7.38 PBS.

subtracted). Upon further treatment of the sensing interface with the second amplification probes, i.e. the colloidal gold immunocomplex, a substantially increased absorption band was obtained. As is shown in Fig. 2, curve c, one observes

that the absorption band gives a maximum about 0.07 (the blank absorbance was subtracted), implying that the two-step amplification procedure offered an enhanced signal as 3.5 times large as the primary sandwich amplification step did. The mass amplification of the colloidal gold immunocomplex was further confirmed by a TEM investigation of the gold nanoparticles and the colloidal gold immunocomplex. It can be seen from Fig. 3 that the gold nanoparticles are nearly monodispersed, while the colloidal gold immunocomplex are organized in big aggregates with similar distances separating the individual nanoparticles from one another. This gives the microscopic evidence that protein A interacts with antibody on the colloidal gold surface, mediating the aggregation of nanoparticles and the formation of dendritic colloidal gold immunocomplex. 3.3. Optimization of the dilution ratio of antibody–colloidal gold conjugate

Fig. 2. Absorption spectra of a glass support: (a) after modification with goat anti-human IgG antibody according to Scheme 1; (b) after interaction of the antibody-functionalized glass with human IgG and then with the antibody–colloidal gold conjugate; (c) after the second amplification step with the colloidal gold immunocomplex.

Fig. 4 shows the effect of the dilution ratio of the antibody–colloidal gold conjugate on the sensing response of human IgG. It is observed from Fig. 4, curve a, that the primary amplification signal resulting from the adsorption of the antibody–colloidal gold conjugate increases with

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increase until the antigen sites reactive to the antibody are saturated. Nevertheless, it seems that the adsorption of excess antibody–colloidal gold conjugate on the electrode surface leads to a tightly packed antibody layer, which might generate great steric hindrance for the subsequent association with the secondary amplification probe, i.e. the dendritic colloidal gold immunocomplex, inducing a decrease of the secondary amplification signal. As a matter of fact, as is observed in Fig. 4, curve b, when the dilution ratio of the colloidal gold-labeled antibody is smaller than 2.5, the second amplification step gives a substantial loss in the frequency response. Therefore, a dilution ratio of 2.5-fold was selected for the further studies. 3.4. Optimization of the component ratio of colloidal gold immunocomplex

Fig. 3. TEM images (100,000×) of: (a) gold nanoparticle suspension and (b) colloidal gold immunocomplex of antibody-functionalized gold nanoparticles and protein A-functionalized gold nanoparticles.

the decrease of the dilution ratio, i.e. the increase of the concentration of the colloidal gold-labeled antibody. This result seems quite immediate, since the amount of adsorbed antibody–colloidal gold conjugate will always

Fig. 4. Effect of the dilution ratio of the antibody–colloidal gold conjugate on the amplification signal: (a) the primary amplification signal; (b) the secondary amplification signal. The frequency decrease values were measured in air. Error bars represent S.D., n = 4.

The effect of the component ratio of the colloidal gold immunocomplex on the secondary amplification signal was investigated. The ratio between the colloidal gold-labeled antibody and the colloidal gold-labeled protein A in the preparation of the colloidal gold immunocomplex plays an important role in determining the sensitivity enhancement in the dendritic secondary amplification step. When the colloidal gold-labeled antibody is excessive, the exterior of the colloidal gold immunocomplex will be surrounded by colloidal gold-labeled antibody, implying that there exists very great steric hindrance for the interaction between the immunocomplex and the sensing interface functionalized with the primary amplification probe. Therefore, the dendritic colloidal gold immunocomplex cannot interact efficiently with the electrode surface, leading to an insignificant signal in the secondary amplification step. On the contrary, when the colloidal gold-labeled protein A is added in an excessive amount, the exterior of the colloidal gold immunocomplex will be surrounded by the colloidal gold-labeled protein A, providing reactive sites to interact with the sensing interface functionalized with the primary amplification probe. However, when the colloidal gold-labeled protein A is too excessive, one might infer that these excessive ligands to the colloidal gold-labeled antibody will result in a complex with only one layer of colloidal gold-labeled protein A surrounding a antibody-functionalized gold nanoparticle, precluding the formation of big dendritic colloidal gold complex and further enhancement of the sensitivity. Therefore, a proper ratio of the colloidal gold-labeled protein A to the colloidal gold-labeled antibody is very essential for the formation of dendritic colloidal gold complex with appropriate size and thus contributes the optimum mass amplification factor. As is shown in Fig. 5, when the volume ratio of the colloidal goldlabeled antibody to the colloidal gold-labeled protein A is 1.2, the maximum secondary amplification signal is obtained. Therefore, the volume ratio of the colloidal gold-labeled antibody to the colloidal gold-labeled protein A was selected to be 1.2 in the preparation of the colloidal gold complex in further studies.

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Fig. 5. Effect of the component ratio of the colloidal gold immunocomplex on the secondary amplification signal. The frequency decrease values were measured in air. Error bars represent S.D., n = 4.

3.5. Performance of the immunosensor with dendritic amplification The frequency changes resulting from the sensing of the analyte IgG of different concentrations are shown in Fig. 6. The primary amplification and the secondary dendritic amplification procedures both enabled the sensing of the analyte in the concentration range from 10.9 ng ml−1 to 10.9 ␮g ml−1 with a saturated frequency response obtained at the concentration above 10.9 ␮g ml−1 . In the absence of the amplification steps, frequency shift response of only −15 Hz was detected for the antigen–antibody recognition event at the IgG concentration as high as 10.9 ␮g ml−1 . However, a human IgG solution with a concentration of 10.9 ng ml−1 was detected immediately using these two amplification procedures, and one observed frequency responses of −57 and

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−140 Hz, respectively, for the primary amplification step and the secondary amplification one. At a concentration of 10.9 ␮g ml−1 of the analyte IgG, the crystal frequency changes due to the primary amplification and the secondary amplification were −195 and −494 Hz, respectively, indicating that the signal amplification of the primary and the secondary amplification process was about 13- and 33-folds, respectively. According to the three S.D. rule (S.D. denotes the standard deviation of five measurements of a blank solution), the detection limit of the primary and the secondary amplification procedures was estimated to be 9.7 and 3.5 ng ml−1 , respectively, while that for direct sensing of the analyte IgG without amplification was 10 ␮g ml−1 . This result implied that the sensitivity and the detection limit of the introduced dendritic amplified sensing was dramatically improved compared to the direct QCM-based immunosensor.

4. Conclusion A dendritic immunocomplex of protein A-functionalized Au nanoparticles and antibody-functionalized Au nanoparticles was demonstrated as a novel mass amplification probe for microgravimetric QCM-based immunosensing. Coupled with a protein A-based oriented immobilization of the antibody, the presented dendritic amplification procedure was shown to offer significant enhancement in the sensing sensitivity and was capable of lowering the detection limit by three orders of magnitude with comparison to the direct QCM determination without amplification. This type of assay does not require any commercial label reagents, and can be implemented with ease and low cost. Nevertheless, the suggested assay procedure requires relatively prolonged incubation steps compared to the simple direct assay, indicating this method is not suitable for rapid detection applications. It is expected that this QCM-based immunosensing method might be a cost-efficient and convenient alternative to other high sensitive optical immunoassay techniques in cases which the detection sensitivity is of dominant significance in comparison with the analysis time.

Acknowledgement Financial support from the National Natural Science Foundation of China (Grant Nos. 20105007) is gratefully acknowledged.

Fig. 6. Frequency changes of the antibody-immobilized Au-quartz crystal upon sensing of the analyte IgG of different concentrations: (a) upon the association of the analyte IgG with the sensing interface; (b) upon the primary amplification of the colloidal gold-labeled antibody with the resulting interface; (c) upon the dendritic amplification of the colloidal gold immunocomplex with the resulting interface. The frequency decrease values were measured in air. Error bars represent S.D., n = 4. The data shown at the left of the breakers are the frequency decreases measured on the blank solution.

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Biographies Xia Chu, Ph.D., is an associate professor of chemistry in College of Chemistry and Chemical Engineering, Hunan Normal University. Her research interest covers biosensors and chemical sensors. Zi-Long Zhao was graduated from Hunan Normal University in 2003 majoring in chemistry. He is currently a graduate student of Hunan Normal University. Guo-Li Shen is a professor of chemistry in College of Chemistry and Chemical Engineering, Hunan University, Changsha, China. He is the vice-chairman of the Chemical Sensor Subcommittee of Chinese Analytical Instrumentation Society and acting deputy editor-in-chief of ‘Chemical Sensor’. He was graduated from Department of Chemistry, Fudan University, Shanghai in 1961. His research interest covers chemical and biosensors. Ru-Qin Yu is a professor of chemistry, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China. He has been a member of Chinese Academy of Sciences since 1991. He is the editorin-chief of ‘Chemical Sensor’, editorial adviser of Analytical Chimica Acta (Elsevier) and Journal of Chemometrics (Wiley). He was graduated from Department of Chemistry, St. Petersburg University, Russia in 1959. His research interest covers chemical sensors and chemometrics.