A cost-effective sandwich electrochemiluminescence immunosensorfor ultrasensitive detection of HIV-1 antibody using magneticmolecularly imprinted polymers as capture probes

Jing Zhou a, Ning Gan a,n, Tianhua Li a, Futao Hu b, Xing Li a, Lihong Wang c, Lei Zheng d

a State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University,Ningbo 315211, PR Chinab Faculty of Marine, Ningbo University, Ningbo 315211, PR Chinac Faculty of Science, Ningbo University, Ningbo 315211, PR Chinad Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China

a r t i c l e i n f o

Article history:Received 17 September 2013Accepted 24 October 2013Available online 9 November 2013

Keywords:Sandwich electrochemiluminescenceimmunosensorSurface imprintingEpitope imprintingHuman immunodeficiency virus type1 antibodyHorseradish peroxidaseLuminol

a b s t r a c t

In this report, a rapid and cost-effective sandwich electrochemiluminescence (ECL) immunosensor wasconstructed for the ultrasensitive detection of human immunodeficiency virus type 1 antibody (anti-HIV-1)using magnetic molecularly imprinted polymers (MMIPs) as capture probes by combining surface andepitope imprinting techniques and antigen conjugated with horseradish peroxidase (HRP-HIV-1) aslabels. First, 3-aminobenzeneboronic acid (APBA) was used as the functional monomer and cross-linkingreagent, which was polymerized on the surface of silicate-coated magnetic iron oxide nanoparticles(Fe3O4@SiO2 NPs) in the presence of human immunoglobulin G (HIgG), as the template exhibiting thesame Fc region but different Fab region to anti-HIV-1 after the addition of the initiator, ammoniumpersulfate. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film onthe Fe3O4@SiO2 NPs. Thus, MMIPs, which could be reused after eluting the template, were used torecognize and enrich ultra-trace levels of anti-HIV-1. Subsequently, a novel sandwich ECL immunosensorwas formed through the immunoreaction between MMIPs conjugated with varied concentrations ofanti-HIV-1 and HRP-HIV-1. By the catalysis of HRP immobilized onto HRP-HIV-1 on the ECL system ofLuminol-H2O2, a linear response range of the anti-HIV-1 dilution ratio (standard positive serum) wasachieved from 1:20,000 to 1:50, with a detection limit of 1:60,000 (S/N¼3). The developed methodprovides a low-cost, simple, and sensitive way for the early diagnosis of HIV infected patients.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Acquired immune deficiency syndrome (AIDS) is caused byinfection with human immunodeficiency virus (HIV), and is theend-stage of disease (Weiss, 1993). HIV is divided into two types,that is, HIV-1 and HIV-2, of which HIV-1 is more common andextensively studied (Wang et al., 2013; Jangam et al., 2013;Esseghaier et al., 2013; Ruslinda et al., 2013). To date, no drug orvaccine has become available to cure or stop the onset of disease(Garber et al., 2004; McMichael, 2006; Levy, 2009; Ross et al., 2010).The early diagnosis and monitoring of HIV-1 infection in patientsis of great importance for the initiation and the evaluation ofantiviral therapies (Cohen et al., 2011).

HIV-1 antibody (anti-HIV-1) is generated after HIV-1 infection(Ly et al., 2001). However, the window of opportunity to detectanti-HIV-1 is generally 4–8 weeks (Weiss, 1993), which causes great

difficulty in the early diagnosis of HIV infection. Therefore, variousmethods have been developed for the early detection of anti-HIV-1 and reduce the diagnostic window, hoping to find thepotential opportunity of a cure in the early stages of infection(Ly et al., 2001).

At present, these methods mainly include enzyme-link immu-nosorbent assay (ELISA) (Louie et al., 2006; Yeom et al., 2006),enzyme immunoassay (EIA) (Barbe et al., 1994; Galli et al., 1996),and Western Immunoblot (WB) analysis (Dodd and Fang, 1990).Although these methods offer a highly accurate approach in thetesting of anti-HIV-1, their low sensitivity makes the diagnosticwindow lag behind. In this case, there would be no opportunity tocure this disease after anti-HIV-1 was detected (Cohen et al., 2008).Thus these methods are not suitable for the reduction in thediagnostic window of opportunity. To address this problem andobtain a rapid diagnosis, developing new methods for the detec-tion of anti-HIV-1 to improve sensitivity is a key step.

Recently, electrochemiluminescence (ECL) has received a greatdeal of interest in many fields due to its unique advantages, suchas an innately high sensitivity, simple instrumentation and low

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bios.2013.10.044

n Corresponding author. Tel./fax.: þ86 574 87609987.E-mail address: ganning@nbu.edu.cn (N. Gan).

Biosensors and Bioelectronics 54 (2014) 199–206

cost, including environmental pollutant determination, pharma-ceutical analysis, and immunoassays (Richter, 2004; Zhang et al.,2008; Jia et al., 2009; Hull et al., 2009; Wu et al., 2010; Zhao andZhou, 2012; Carvajal et al., 2012; Dai et al., 2012). Even so, itcommonly needs the use of magnetic beads to label the antibodyor antigen as the capture probes for commercial ECL immunoas-says (ECLIA). This makes the labeling steps cumbersome whenpreparing such probes. Moreover, the prepared probes are difficultto preserve and easily inactivated, which directly affects thesensitivity of ECL detection and increases the testing costs.

With the development of molecular imprinting technology,molecularly imprinted polymers (MIPs) have been assessed asartificially synthesized receptors and widely used in sensors,catalysis reactions, and separations, in which MIPs act as sub-stitutes for antibodies or enzymes due to their unique bindingcharacteristics (in terms of both affinity and specificity), highchemical and physical stability, easy availability and low cost(Haupt and Mosbach, 2000; Ye and Mosbach, 2008). However,imprinting of large structures, such as proteins and other bioma-cromolecules, remains a challenge, and is attributable to theirrestricted mobility within highly cross-linked polymer networks,and the poor efficiency in rebinding. Meanwhile, the molecularsize, conformational flexibility and sensitivity to denaturation ofproteins would also make them difficult for imprinting (Lu et al.,2012a). Therefore, some approaches involving surface imprinting(Nematollahzadeh et al., 2011) and epitope imprinting (Tai et al.,2010) have been developed to help solve these issues. Recently,Lu et al. reported a new magnetic surface imprinting techniquebased on Fe3O4@SiO2 NPs as the core (Lu et al., 2012b). The MIPswere coated on Fe3O4@SiO2 NPs, and the resulting polymer wasmagnetically susceptible and thus easily separated by externalmagnetic fields after completing both adsorption and recognition.Moreover, a mass of recognition sites were situated at the surfaceof the core–shell material, which made the templates rebind fasterand removed easily due to easy accessibility and low mass transferresistance to the target molecules. Furthermore, the MMIPs alsodisplay a high adsorption ability and excellent adsorption selec-tivity. These advantages of MMIPs make this approach attractiveand broadly applicable to biological enrichment, separation andbiosensors (Kan et al., 2010). On the other hand, epitope imprint-ing has been an important area of research since it was firstdemonstrated by Minoura et al. for peptide recognition (Rachkovand Minour, 2000). In this process, a fragment exposed on theepitope of the target macromolecule is used as a template. Theresultant MIP recognizes not only the template but also the wholemacromolecule. For example, Shea et al. constructed macromolecu-lar receptors for proteins using the epitope-peptides of cytochromeC and bovine serum albumin as templates (Nishino et al., 2006).Recently, Lu et al. developed a biomimetic sensor for the detectionof HIV-1 related protein (glycoprotein 41, gp41) based on anepitope imprinting technique (Lu et al., 2012a). Compared withtraditional protein imprinting approaches, epitope imprinting hasseveral advantages (Ge and Turner, 2008). Firstly, more specificand stronger interactions with a fragment or small part of themacromolecule can reduce non-specific binding and improveaffinity. Secondly, the polymer can recognize both the templateand the entire protein, and the operational procedures are easierto complete. Thirdly, short peptides as epitopes for imprinting arecost-effective. However, little work has been reported thus far oncombining surface imprinting with epitope imprinting for detec-tion of proteins in human samples.

Based on the above-mentioned factors, a novel sandwich ECLimmunosensor for the ultratracible detection of anti-HIV-1 wasdesigned using MMIPs as an alternative to HIV-1 antigens ascapture probes by combining surface and epitope imprintingtechniques and using HRP-HIV-1 as labels. The whole process for

the construction of the immunosensor is shown in Scheme 1.Since HIgG bears the same Fc region, and a different Fab region toanti-HIV-1, it can be used as the dummy template for MMIPs inplace of anti-HIV-1. Moreover, it is much cheaper, more availablethan anti-HIV-1 and the resulting MMIPs can be reused aftereluting the template, all of which make the immunoassay cost-effective. Furthermore, the obtained MMIPs also simplified thefabrication process of the immunosensor by the use of externalmagnetic fields. By the catalysis of HRP immobilized onto HRP-HIV-1 and detected by the ECL system of Luminol-H2O2, a linearresponse range of anti-HIV-1 dilution ratios (standard positiveserum) was achieved from 1:20,000 through 1:50 with a detectionlimit of 1:60,000 (S/N¼3). This method associated the recognitionproperty of MMIPs towards a target molecule with the highspecificity of an antibody-antigen reaction, which provided animproved specificity when compared with MMIPs alone. In addi-tion, the features of this improved method including its low cost,simplicity, and ultrasensitivity, would pave the way for a newapproach in clinical immunoassay.

2. Experiments

2.1. Chemicals and reagents

HIV ELISA Kits were from Rongsheng Biological PharmaceuticalCo. Ltd. (Shanghai, China). Elecsys HIV Kits were from RocheDiagnostics GmbH., Germany. Human immunoglobulin G (HIgG)was from Beijing Biosynthesis Biotechnology Co. Ltd. TetraethylOrthosilicate (TEOS), 3-aminobenzeneboronic acid (APBA) wasfrom J&K chemical Co. Ltd. Luminol was from Sigma-Aldrich(St. Louis, MO, USA). Bovine serum albumin (BSA, 96–99%) and TritonX-100 (TX-100) were bought from sinophram chemical reagent Co.Ltd. (Shanghai, China). Other chemicals were analytically pure andused as received. A 0.01 M stock solution of luminol was preparedby dissolving luminol in 0.1 M NaOH solution and was kept at 4 1C.Working solutions of luminol were obtained by diluting the stocksolution. 0.1 M phosphate buffer solutions (PBS, pH 8.0) containing0.1 M KCl and NaCl was used as the electrolyte. Washing buffersolution consisted of a PBS (pH 7.4) with 0.05% (v/v) Tween 20(PBST). Eluting solution contained 1% sodium dodecyl sulfate(SDS). Deionized water was used throughout the experiments.

2.2. Instrumentation

ECL experiments were carried out using a MPI-B modelelectrochemiluminescence analyzer (Xi'an Remax ElectronicScience&Technology Co. Ltd., Xi'an, China) with the voltage ofthe photomutiplier tube (PMT) set at 600V. A three-electrodesystem used consisted of a screen-printed carbon working elec-trode (SPCE, DropSens Corporation, Spain), a carbon auxiliaryelectrode and an Ag reference electrode. The developed assaywas validated using a commercialized electrochemiluminescenceimmunoassay (ECLIA) on the fully automated immuno-analyzerElecys 2010 (Roche). A field emission-scanning electron micro-scope (FE-SEM, SU-70, Hitachi, Japan), and transmission electronmicroscope and high resolution transmission electron microscope(TEM and HRTEM, Tecnai G20, Philip) were employed to char-acterize the surface morphology and size of the nanoparticles.Atomic force microscopic (AFM) images were obtained by aNanoscope IIIa multimode atomic force microscope (Veeco Instru-ments, USA). The XRD characterization was performed using X-raydiffraction (Bruker, D8 Focus) with Cu Kα radiation at roomtemperature. Thermogravimetric analysis (TGA) was carried outby a ZRY-2P thermal analyzer (Shanghai Balance Instrument

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206200

Company, China). Magnetic properties were obtained with aLakeShore 7307 (Lakeshore Cryotronic) VSM at 300 K.

2.3. Preparation of MMIPs and the ECL immunosensor

MMIPs and magnetic non‐molecular imprinted polymers (MNIPs)were prepared according to a slightly modified report (Li et al., 2009).Firstly, an aqueous suspension of superparamagnetic magnetitenanoparticles were prepared by the controlled chemical coprecipita-tion reaction. FeCl2 �4H2O (3.44 g) and FeCl3 �6H2O (9.44 g) wererespectively dissolved under a N2 atmosphere in deaerated deionizedwater (160 mL) with vigorous mechanical stirring (800 rpm). Anitrogen gas environment was maintained in the vessel during thereaction to prevent critical oxidation. When the solution was pre-heated to 80 1C, ammonium hydroxide (20 mL) was added to achievealkaline conditions. After 30 min, black superparamagnetic MNPs

were obtained by sedimentation with the help of an externalpermanent magnet and the supernatant was decanted. The MNPswere washed with deionized water and absolute ethanol severaltimes (150 mL each time) to remove unreacted chemicals until atransparent ferrofluid was obtained. Then, the superparamagneticMNPs were coated with silica by using a sol–gel method. Super-paramagnetic MNPs (0.120 g) was redispersed in 2-propanol(240 mL) and deionized water (18 mL) by sonication for approxi-mately 15 min. Then, under continuous mechanical stirring(800 rpm), ammonium hydroxide (21 mL) and TEOS (4 mL) wereconsecutively added to the reaction mixture. The reaction proceededat room temperature for 14 h under continuous mechanical stirring.The resultant product was obtained by magnetic separation withthe help of an external permanent magnet and was thoroughlywashed with deionized water and absolute ethanol. Lastly, for thepreparation of human IgG-imprinted polymers (HIgG-MMIPs), HIgG

Scheme 1. Describing the systematic process for the synthesis of the immunosensor.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206 201

(0.01 g) was dissolved in sodium phosphate buffer (5 mL, pH 8.0)containing APBA (100 mM), and the mixture was incubated atroom temperature for 1 h. After adding silica-coated MNPs(0.04 g), the solution was then incubated for 2 h at room tem-perature. Prior to use, the silica-coated MNPs were subjected toextensive deionized water and absolute ethanol, and washedthoroughly. Subsequently, a 100 mM aqueous solution of ammo-nium persulfate (6.5 mL) as initiator was slowly added dropwise tothe above solution for about 20 min and the polymerizationprocess was executed at room temperature. After 14 h, HIgG-MMIPs were obtained. Finally, the HIgG-MMIPs were eluted with1% SDS to remove the entrapped HIgG. The MMIPs were thenequilibrated with the buffer used in the polymerization process.MNIPs were prepared using the same procedure but without HIgG.The procedures are shown in Scheme 1A and B.

The ECL immunosensor was constructed as Scheme 1B: 10 μL10 mg mL�1 MMIPs and 20 μL different dilution ratio of anti-HIV-1 solution (1:20,000 to 1:10) was firstly mixed, adsorbing andstirring for 60 min at 25 1C. Then 50 μL 2.5% BSA was added toprevent the nonspecific adsorption. After 1 h, the mixture wasseparated by magnet and washed twice with water, dispersinginto 40 μL HRP-HIV-1 conjugates and incubating at 37 1C for30 min. Finally, the sandwich-type immunocomplexes were iso-lated by magnet, washed with PBST three times, dispersed in20 μL of PBS (pH 8.0) and stored at 4 1C for ECL tests.

2.4. ECL detection

As shown in Scheme 1C, 10 μL aforementioned immunocom-plexes was dropped on the SPCE with a magnet pasted in advance,then ECL detection could be performed by applying a double-steppotential (10 s pulse period, 1 s pulse time, 0.3 V pulse potentialand 0.15 V initial pulse potential) to the working electrode at ascan rate of 100 mV s�1 after adding 5 μL 2�10�4 M luminolsolution and 10 μL 5�10�3 M H2O2 solution. The resulting ECLsignals from different concentration of anti-HIV-1 solution weredetected by the PMT below. Finally, standard curves were obtainedaccording to the linear relationship between the logarithm of theΔECL intensity IΔECL (IΔECL¼ I� IO, here I and IO are the ECLintensities of ECL immunosensor prepared with and withoutanti-HIV-1, respectively) and the logarithm of the dilution ratioof anti-HIV-1 in the range of 1:20,000 to 1:50.

2.5. ECL adsorption measurement

To investigate the adsorption dynamics of MMIPs and MNIPs,10 μL 10 mg mL�1 MMIPs and MNIPs were separately added into30 μL anti-HIV-1 solution with a dilution ratio of 1:10, then theoptimal adsorbed time was obtained through measuring thechanges in ECL intensity, ΔECL of ECL immunosensor preparedat different time intervals. To investigate the adsorption equili-brium of MMIPs, 10 μL 10 mg mL�1 MMIPs was equilibrated withvaried initial different dilution ratio of anti-HIV-1 solution(1:10,000–1:10). After 60 min, the polymers were separated bymagnet and the saturated adsorption capacity Q0 on MMIPs wasassessed by ΔECL at adsorption equilibrium.

3. Results and discussion

3.1. Characterization of synthesized nanoparticles

The SEM and TEM images of Fe3O4, Fe3O4@SiO2 and MMIPsnanocomposites are shown in Fig. 1. The diameters of theuncoated Fe3O4 nanoparticles were in the range of 10–18 nm(Fig. 1A and D), while the mean diameter of the Fe3O4@SiO2

nanoparticles increased to approximately 100 nm with a narrowsize distribution (Fig. 1B and E). The latter size was determined bythe TEOS concentration in the reaction mixture. It was also shownthat the Fe3O4 nanoparticles were fully coated by the silica(Fig. 1E). After the imprinted polymer further covered the Fe3O4@-SiO2 nanoparticles, the surface of the nanoparticles became lesssmooth, suggesting the successful formation of imprinted layers(Fig. 1C and F). The HRTEM was used to further confirm theformation of the protein-templated imprinted sites. It could beseen from Fig. 1G that there existed many cavities on the surface ofMMIPs. AFM was also used to characterize the surface morphologyof the imprinted layers before (Fig. 1H) and after (Fig. 1I) theelution of the HIgG templates. Each scan represents a 200 nm�200 nm lateral area and the vertical scale is 2.8 nm per division.Compared with Fig. 1H, I showed many dark points whose lateralareas were about (6–12) nm� (8–15) nm and depths were in therange of 0.5–1.5 nm, indicating the formation of the cavities on theimprinted layers.

Fig. 1J showed XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b) andMMIPs (c). It was observed that the six diffraction peaks of Fe3O4

(2θ¼30.11, 35.51, 43.11, 53.41, 57.01, 62.61) appeared in the 2θrange of 20–801. After Fe3O4 was coated by SiO2 NPs, the peakintensity decreased and a new parabolic peak attributed toamorphous SiO2 NPs appeared at approximately 2θ¼231. Finally,from curve c, it was observed that the peak intensity of MMIPs wasslightly reduced when compared with that of Fe3O4@SiO2 NPs,which revealed the formation of a thin imprinted film. However,the peak positions of Fe3O4, Fe3O4@SiO2 and MMIPs wereunchanged, demonstrating that their crystalline structure wasessentially maintained.

Fig. S1 (please refer to the Supporting information) essentiallycompared the TGA curves of Fe3O4@SiO2 and MMIPs, whichillustrated that the weight loss of curve a, and curve b below130 1C (about 9%) was due to volatilization of the solvent or water.The vast and rapid weight loss of curve b between 300 1C and620 1C (about 30%) originated from the imprinted polymer on thesurface of Fe3O4@SiO2. Therefore, these observations indicated theexistence of an imprinted polymer.

VSM was employed to study the magnetic properties of thesynthesized nanocomposites: Fe3O4 (a), Fe3O4@SiO2 (b), andMMIPs (c). The magnetic hysteresis loops of the samples dried at300 K are illustrated in Fig. 2. The saturation magnetization ofMMIPs was reduced to 8.572 emu g�1 as compared with bulk Fe3O4

and Fe3O4@SiO2 (saturation magnetization are 58.921 emu g�1

and 10.388 emu g�1, respectively). However, the saturation mag-netization of MMIPs retained sufficient magnetic responsivenessto satisfy the need of the magnetic separation (Fig. 2 (inset)).When an external magnetic field was applied, the dark particleswere attracted to the wall of the vial in a very short time (about20 s) and the dispersion became transparent and clear. Afterremoving the external magnetic field, a brownish black homo-geneous dispersion appeared. The results demonstrated the super-paramagnetism property of MMIPs.

3.2. Characterization of the ECL immunosensor

As shown in Fig. 3, no significant difference in the ECL signalcould be found in MNIPs (curve a) and MMIPs (curve b) for the ECLimmunosensor when there was no anti-HIV-1 in sample. However,when there existed anti-HIV-1 in sample, the ECL intensities fromMNIPs (curve c) and MMIPs (curve d) immunosensor were bothenhanced because the immobilization of HRP on them couldcatalyze the ECL reaction of the luminol-H2O2 system. Addition-ally, the signal originating from MMIPs was stronger as comparedwith that of MNIPs, which was ascribed to a higher specificadsorption capacity of anti-HIV-1 on MMIPs.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206202

3.3. Optimization of the experimental conditions

In order to enhance the detection sensitivity of the sandwichimmunosensor and reduce background signals, we optimized someconditions including the amount of MMIPs, and the pH of the

electrolyte, which was achieved by changing the examined condi-tions while the others were fixed.

The effect of the amount of MMIPs used to construct thesandwich immunosensor on the sensitivity of the ΔECL signal wasinvestigated from 20 to 200 μg (Fig. S2, Supporting information).

Fig. 1. The SEM images of Fe3O4 (A), Fe3O4@SiO2 (B) and MMIPs (C); TEM images of Fe3O4 (D), Fe3O4@SiO2 (E) and MMIPs (F); HRTEM images of MMIPs (G); AFM imagesof the imprinted layers before (H) and after (I) the elution of the HIgG templates; (J) XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b) and MMIPs (c).

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206 203

An increase in the ΔECL signal was observed up to 100 μg.Whereas, the increasing amount of MMIPs lowered the sensitivityfor ECL detection, which was probably due to an excess of MMIPsblocking the mass transformation, and minimizing the electricconductivity. For this reason, an immobilized MMIPs, at an amountof 100 μg was chosen for further work. Since the luminol-H2O2

system in base solution could lead to a strong ECL backgroundsignal, 2�10�4 M was selected as the concentration of luminolsolution in this study. By contrast, to ensure sufficient signalamplification of HRP from the signal tags HRP-HIV-1 and pre-servation of HRP activity, 5�10�3 M H2O2 solution and pH 8.0 PBSas the electrolyte were thus adopted in this paper (Fig. S3, Supportinginformation).

3.4. Imprinting effect

The adsorption kinetics (Fig. 4a) and isotherm curves (Fig. 4b)of MMIPs and MNIPs are shown herein. It could be seen thatMMIPs achieved a much higher ΔECL and a faster adsorption ratethan MNIPs during the first 60 min and until adsorption equili-briumwas achieved (Fig. 4a). However, it should be noticed MNIPsreached adsorption equilibrium much faster (40 min) than MMIPs(60 min), which might be attributed to the higher adsorptioncapacity of MMIPs and the need of more adsorption time. Butthe purpose of this paper is to develop an ultrasensitive ECLimmunosensor and reduce the diagnostic window between thetime of HIV-1 infection and laboratory diagnosis. Thus improvingthe detection sensitivity and reducing the detection limit is thekey to this work. To achieve the goal, what is more important isthe higher adsorption of MMIPs, which contributes to the enrich-ment of ultratrace analytes. As presented in Fig. 4b, the ΔECLincreased as the initial dilution ratio of anti-HIV-1 increased atdilution ratios below 1:50, and reached a plateau at a dilution ratioof 1:50. Moreover, the MMIPs exhibited a higher ECL change foranti-HIV-1 than MNIPs did at the same concentration. Thisindicated specific adsorption of MMIPs and the non-specificbinding of MNIPs within the range of the experimental concentra-tions. By fitting the experimental data with the isothermicadsorption curves, the saturated adsorption capacity Q0 of MMIPswas approximately 0.006 C0 g�1 (where C0 was the original con-centration of anti-HIV-1 for standard positive serum).

3.5. Analytical performance

The ECL signals that were responsive to the changing dilutionratio of anti-HIV-1 (a–j) are shown here (Fig. 5). It could be seenthat the ECL peak intensity increased gradually with increasing

Fig. 2. The hysteresis loops of Fe3O4 (a), Fe3O4@SiO2 (b), and MMIPs (c). The insertshows the separation and redispersion process of a solution of MMIPs in thepresence (left) and absence (right) of an external magnetic field.

Fig. 3. ECL curves of a mixture included 5 μL 2�10�4 M luminol solution, 10 μL5�10�3 M H2O2 solution and 10 μL ECL immunosensor prepared by MNIPs andHRP-HIV-1 without (a) and with (c) anti-HIV-1 at a dilution ratio of 1:50;additionally, 5 μL 2�10�4 M luminol solution, 10 μL 5�10�3 M H2O2 solutionand 10 μL ECL immunosensor prepared by MMIPs and HRP-HIV-1 without (b) andwith (d) anti-HIV-1 at a dilution ratio of 1:50.

Fig. 4. (a) Adsorption kinetics of MMIPs and MNIPs; (b) Adsorption isothermic curves of MMIPs and MNIPs.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206204

concentrations of anti-HIV-1 (b–j). The standard calibration curvefor anti-HIV-1 detection is shown in the inset. Under optimalconditions, the logarithm of the ΔECL intensity IΔECL (IΔECL¼ I� IO,here I and IO are the ECL intensities of ECL immunosensor preparedwith and without anti-HIV-1, respectively) was proportional to thelogarithm of the dilution ratio of anti-HIV-1 in the range of1:20,000 to 1:50 (R2¼0.9927), and the detection limit was1:60,000 (S/N¼3), which was much lower than that found forELISA (1:300) (Saifuddin et al., 1995). This result indicated that theemergence of MMIPs as a candidate for biological recognitionelements and signal amplification would offer new perspectiveson the improvement of current immunoassays.

3.6. Specificity, reproducibility and feasibility of this proposedimmunosensor

To study the specificity of this proposed immunosensor, MMIPswere binded respectively with anti-HIV-1 standard negative serum(SNS), 20 ng mL�1 carcinoembryonic antigen (CEA), 10 ng mL�1

carbohydrate antigen 19-9 (CA19-9), 1 mg mL�1 BSA, 1 μg mL�1

HIgG, 100 ng mL�1 alpha fetoprotein (AFP), anti-HIV-1 with a dilu-tion ratio of 1:2000 and a mixture containing all the abovemolecules/proteins, blocked, then incubated with HRP-HIV-1. Thesubsequent detection was followed by using the current immuno-sensor procedure (Fig. S4, Supporting information). The ECL responseobtained from MMIPs binding with anti-HIV-1 and incubated withHRP-HIV-1 was much stronger than that obtained from MMIPsbinding with SNS, CEA, CA19-9, BSA, HIgG and AFP, and incubatedwith HRP-HIV-1, which was similar to that obtained with MMIPsbinding with a mixture containing all of the above molecules/proteins and incubated with HRP-HIV-1. These results indicated thatthis immunosensor had adequate specificity for the diagnosis ofanti-HIV-1.

The reproducibility of the developed immunosensor wasinvestigated in the online assay for anti-HIV-1 with a dilutionratio of 1:500. High reproducibility of the ECL response (relativestandard deviation (RSD) of 4.52%) was obtained for 5 repetitivedetections of anti-HIV-1 with a dilution ratio of 1:500. Theelectrode-to-electrode reproducibility was also examined betweensix independent SPCE electrodes in the above solutions, and thecorresponding RSD was calculated to be 3.89%. In addition, theavailable times of MMIPs eluting from the sandwich immunosen-sor were studied by repetitive fabrication of the immunosensorand detection of the ECL signal. The observation informed us that

there was no significant difference in ECL signals observed for tenmeasurements, which might be explained by the excellent stabi-lity of MMIPs.

To monitor the feasibility of the developed immunosensor inclinical analyses, three blank serum samples from differentpatients were diluted and analyzed. Recovery experiments werecarried out by the standard addition method in these serumsamples. The results showed that the recovery ranged from84.15% to 104.50%, and that the corresponding RSD ranged from4.28% to 8.31% (see Supporting information, Table S1), whichrevealed that the feasibility of the developed immunoassay couldbe preliminarily applied to the detection of ultratrace levels ofanti-HIV-1 in clinical human serum samples for routine clinicaldiagnosis. In addition, to further validate the proposed method,forty-three human serum samples from Nanfang Hospital (China)were tested with the proposed method and a commercializedECLIA kit (Roche), which were consistent with each other (25samples were positive and the rest were negative). The efficiencyvalue of 100% could be calculated using the followingequation (Shukla et al., 2009): Efficiency¼(TPþTN)�100/Total(TP, True Positive; TP¼25; TN, True Negative; TN¼18; Total¼43).Such result demonstrated that the novel method exhibited goodassay performance comparable to the commercialized ECLIA kitand was potentially applicable in detecting anti-HIV-1.

4. Conclusions

We have successfully designed a rapid and cost-effectivesandwich ECL immunosensor for the ultrasensitive detection ofanti-HIV-1 based on the combination of surface imprinting andepitope imprinting techniques. We have also studied its effective-ness for detecting human serum specimens by ECLIA. The resultingimprinted film showed a high affinity and enrichment capacity foranti-HIV-1 at ultra low concentration levels. Additionally, ECLmeasurements revealed that the developed sandwich ECL immu-nosensor had a stronger capacity of resisting disturbance thanMMIPs alone, owing to the use of the high specificity of anantibody-antigen reaction, which overcame the interference ofthe possible template molecules. The capture probes (MMIPs)could also be recycled, and thus greatly reduced the experimentalcost. Therefore, the obtained immunosensor was applied success-fully for monitoring anti-HIV-1 in human serum samples. Thesimplicity, cost-effectiveness and high sensitivity of the developedmethod proposes a merger of imprinting techniques and immu-noassay for biomolecular analysis.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No. 30901367), the Natural Science Foundationof Zhejiang (LY13C200017, LY12C20004, and LY12B01005), theScience and Technology Project of Zhejiang (2012C23101 and2011C23126), the Natural Science Foundation of Ningbo City(2011B82014 and 2013A610241, and K.C. Wong Magna Fund inNingbo University.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2013.10.044.

Fig. 5. ECL profiles of immunosensor detection of dilution ratios of anti-HIV-1:(a) 0, (b) 1:20,000, (c) 1:10,000, (d) 1:8000, (e) 1:5000, (f) 1:2000, (g) 1:500,(h) 1:200, (i) 1:100, and (j) 1:50. Inset: calibration curve.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206 205

References

Barbe, F., Klein, M., Badonnel, Y., 1994. Ann. Biol. Clin. 52, 341–345.Cohen, M.S., et al., 2011. N. Engl. J. Med. 365, 493–505.Cohen, M.S., Hellmann, N., Levy, J.A., DeCock, K., Lange, J., 2008. J. Clin. Invest. 118,

1244–1254.Carvajal, M.A., Ballesta-Claver, J., Morales, D.P., Palma, A.J., Valencia-Mirón, M.C.,

Capitán-Vallvey, L.F., 2012. Sens. Actuators B Chem. 169, 46–53.Dai, H., Yang, C.P., Tong, Y.J., et al., 2012. Chem. Commun. 48, 3055–3057.Dodd, R.Y., Fang, C.T., 1990. Arch. Pathol. Lab. Med. 114, 240–245.Esseghaier, C., Ng, A., Zourob, M., 2013. Biosens. Bioelectron. 41, 335–341.Galli, R.A., Castriciano, S., et al., 1996. J. Clin. Microbiol. 34, 999–1002.Garber, D.A., Silvestri, G., Feinberg, M.B., 2004. Lancet Infect. Dis. 4, 397–413.Ge, Y., Turner, A.P.F., 2008. Trends Biotechnol. 26, 218–224.Haupt, K., Mosbach, K., 2000. Chem. Rev. 100, 2495–2504.Hull, D.O., Bajrami, B., Jansson, I., Schenkman, J.B., Rusling, J.F., 2009. Anal. Chem. 81,

716.Jangam, S.R., Agarwal, A.K., Sur, K., Kelso, D.M., 2013. Biosens. Bioelectron. 42,

69–75.Jia, T.T., Cai, Z.M., Chen, X.M., Lin, Z.J., Huang, X.L., Chen, X., Chen, G..N., 2009.

Biosens. Bioelectron. 25, 263.Kan, X.W., Zhao, Q., Shao, D.L., Geng, Z.R., Wang, Z.L., Zhu, J.J., 2010. J. Phys. Chem.

B 114, 3999–4004.Levy, J.A., 2009. AIDS 23, 147–160.Li, L., He, X.W., Chen, L.X., Zhang, Y.K., 2009. Chem. Asian J. 4, 286–293.Louie, B., Pandori, M.W., et al., 2006. J. Clin. Microbiol. 44, 1856–1858.Lu, C.H., Zhang, Y., Tang, S.F., Fang, Z.B., Yang, H.H., Chen, X., Chen, G..N., 2012a.

Biosens. Bioelectron. 31, 439–444.

Lu, F.G., Li, H.J., Sun, M., Fan, L.L., Qiu, H.M., Li, X.J., Luo, C.N., 2012b. Anal. Chim. Acta718, 84–91.

Ly, T.D., Laperche, S., Couroucé, A.M., 2001. Eur. J. Clin. Microbiol. Infect. Dis. 20,104–110.

McMichael, A.J., 2006. Annu. Rev. Immunol. 24, 227–255.Nematollahzadeh, A., Sun, W., Aureliano, C.S.A., Lütkemeyer, D., Stute, J., Abdekhodaie, M.

J., Shojaei, A., Sellergren, B., 2011. Angew. Chem. Int. Ed. 50, 495–498.Nishino, H., Huang, C.S., Shea, K.J., 2006. Angew. Chem. Int. Ed. 45, 2392–2396.Rachkov, A., Minour, N., 2000. J. Chromatogr. A. 889, 111–118.Richter, M.M., 2004. Chem. Rev. 104, 3003.Ross, A.L., Bråve, A., Scarlatti, G., Manrique, A., Buonaguro, L., 2010. Lancet Infect.

Dis. 10, 305–316.Ruslinda, A.R., Tanabe, K., Ibori, S., Wang, X.F., Kawarada, H., 2013. Biosens.

Bioelectron. 40, 277–282.Saifuddin, M., Parker, C.J., et al., 1995. J. Exp. Med. 182, 503.Shukla, J., Khan, M., Tiwari, M., Sannarangaiah, S., Sharma, S., Rao, P.V.L., Parida, M.,

2009. Diagn. Microbiol. Infect. Dis 65, 142–149.Tai, D.F., Jhang, M.H., Chen, G.Y., Wang, S.C., Lu, K.H., Lee, Y.D., 2010. Anal. Chem. 82,

2290–2293.Wang, J.H., Cheng, L., Wang, C.H., Ling, W.S., Wang, S.W., Lee, G..B., 2013. Biosens.

Bioelectron. 41, 484–491.Weiss, R.A., 1993. Science 260, 1273–1279.Wu, Y.F., Shi, H.Y., Yuan, L., Liu, S.Q., 2010. Chem. Commun. 46, 7763.Ye, L., Mosbach, K., 2008. Chem. Mater. 20, 959–968.Yeom, J.S., Lee, J.B., et al., 2006. Ann. Clin. Lab. Sci. 36, 73–78.Zhang, J., Qi, H.L., Li, Y., Yang, J., Gao, Q., Zhang, C.X., 2008. Anal. Chem. 80, 2888.Zhao, Z., Zhou, X.M., 2012. Sens. Actuators B Chem. 171–172, 860–865.

J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 199–206206