Analyst

PAPER

Cite this: Analyst, 2016, 141, 3883

Received 31st January 2016,Accepted 19th April 2016

DOI: 10.1039/c6an00254d

www.rsc.org/analyst

Cost-effective and sensitive colorimetricimmunosensing using an iron oxide-to-Prussianblue nanoparticle conversion strategy†

Guanglei Fu,a Sharma T. Sanjaya and XiuJun Li*a,b,c

The development of new sensitive, cost-effective and user-friendly colorimetric bioassays is in increasing

demand to meet the requirement of modern clinical diagnostics and field detection. Herein, a novel iron

oxide-to-Prussian blue (PB) nanoparticle (NP) conversion strategy was developed and applied to sensitive

colorimetric immunosensing of cancer biomarkers. In a typical sandwich-type immunosensing system,

the captured spherical antibody-conjugated iron oxide NPs were transformed into cubic PB NPs, which

exhibited a highly visible blue color with high molar extinction coefficients. Hence, a new colorimetric

immunosensing strategy was developed as a result of this low cost and simple transformation process.

Without the aid of any complex nanoparticle stabilizing ligands and signal amplification processes,

prostate-specific antigen as a model analyte can be detected at a concentration as low as 1.0 ng mL−1 by

the naked eye with good reliability for detection of real human serum samples. This is the first attempt to

develop and apply the iron oxide-to-PB NP colorimetric conversion strategy for immunosensing, and

shows great promise for the development of new sensitive, cost-effective and user-friendly colorimetric

bioassays in various bioanalytical applications, especially in low-resource settings.

Introduction

The “point-of-care” (POC) immunosensing of disease-relatedproteins, especially disease biomarkers, has become increas-ingly desirable to meet the requirement of modern clinicaldiagnostics.1–4 Different analytical principles such as chemi-luminescence, fluorescence, electrochemistry and light scatter-ing have been adopted for immunosensing.5–8 However, thesemethodologies are usually unadaptable for POC diagnostics.One of the most critical bottlenecks for POC detection is theiranalytical readout methods because most readout methodsrequire expensive and bulky instruments and well-equippedlaboratories.9 Attacking this problem head on, the develop-ment of new immunoassays suitable for POC detection isthereby in increasing demand.

Hence, colorimetric assays have been the subject of greatresearch interest because of their incomparable advantages for

POC detection such as simplicity, practicality and ease ofresult readout by only the naked eye.9,10 A variety of nano-materials have been employed as colorimetric probes becauseof their unique physical and chemical properties.11 Amongthose colorimetric probes, noble metal nanomaterials,especially gold nanoparticles (NPs), have been extensively usedin nanoparticle aggregation-based colorimetric assays due totheir high extinction coefficients and distance-dependentoptical properties.12,13 However, gold NPs as colorimetricprobes have some limitations for their wide applicationespecially in resource-limited settings because of their intrin-sic high cost. In order to achieve satisfactory sensitivity, theusage of complex nanoparticle stabilizing ligands and multiplesignal amplification processes are generally indispensable inthese colorimetric assays.9,10,14,15 Additionally, the nondirec-tive properties of nanoparticle aggregation might lead to in-accurate target determination,16 such as a false positiveresponse as a result of the interfering environment-inducednanoparticle self-aggregation.17,18 Hence, the development ofnew, simple, sensitive and cost-effective colorimetric immuno-sensing strategies is of great importance.

With the rapid development of nanoscience, the explorationof nanomaterials with different physical and chemical pro-perties for biosensing provides new opportunities to developnovel colorimetric immunosensing strategies. Prussian blue(PB), an ancient blue dye, is a prototype of mixed-valence tran-

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6an00254d

aDepartment of Chemistry, University of Texas at El Paso, 500 West University Ave,

El Paso, Texas 79968, USA. E-mail: xli4@utep.edubBiomedical Engineering, University of Texas at El Paso, 500 West University Ave,

El Paso, Texas 79968, USAcBorder Biomedical Research Center, University of Texas at El Paso, 500 West

University Ave, El Paso, Texas 79968, USA

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sition metal hexacyanoferrates.19 PB NPs have shown greatpromise for various applications due to their unique electro-chemical, catalytic and magnetic properties.20 In addition totheir low cost, nontoxicity, simple preparation and high struc-tural stability, PB NPs exhibit a highly visible blue color withhigh molar extinction coefficients (1.09 × 109 M−1 cm−1 at808 nm) comparable to that of gold nanorods in the near-infra-red region (700–900 nm),19 providing great potential as apromising colorimetric probe for immunosensing. AlthoughPB NPs have been used in different types of assays such as theelectrochemical sensing and colorimetric detection of glucose,H2O2, vitamin C and hydralazine,21–24 to the best of our knowl-edge, the iron oxide-to-PB NP transformation strategy hasnever been reported for colorimetric immunosensing.Through this conversion strategy, a number of biomoleculescan be detected by this cost-effective and sensitive colorimetricmethod, thus expanding the application of these existingmethods using PB as the colorimetric probe.

Herein, a new colorimetric immunoassay has been pro-posed for sensitive detection of cancer biomarkers using anovel iron oxide-to-PB NP colorimetric conversion strategy asshown in Fig. 1. Using a typical sandwich-type immunoassayas a model system, monoclonal antibody and iron oxide NP-labelled polyclonal antibody are used as the capture antibodyand detection antibody, respectively. After the immunoassayreaction, spherical iron oxide NPs captured in the sandwich-type immunosensing system are dissolved in an acidic solutionto release ferric ions, followed by the reaction with potassiumferrocyanide to produce cubic PB NPs, as shown in Scheme 1.As a result of this novel nanoparticle conversion process, ahighly visible color change is achieved, which enables a newcolorimetric immunosensing method without the aid of anycomplex nanoparticle stabilizing ligands and signal amplifica-tion processes. Using prostate-specific antigen (PSA) as amodel analyte, the performance of the colorimetric immuno-assay and its reliability for PSA detection in real human serumsamples have been systematically studied. As far as we know,this is the first attempt to develop and apply the iron oxide-to-PB NP conversion strategy for colorimetric immunosensing of

cancer biomarkers, which have great potential for the develop-ment of new, sensitive, cost-effective and user-friendly (e.g. noneed of stabilizing ligands and multiple signal amplificationprocesses, and the nontoxic properties of PB) colorimetricbioassays in various bioanalytical and environmental fields.

ExperimentalMaterials

Carboxyl-functionalized iron oxide nanoparticles (NPs) with adiameter of 40 nm were purchased from Ocean NanoTech LLC(USA). Polyclonal rabbit anti-human PSA antibody, monoclonalmouse anti-human PSA antibody and carcinoembryonicantigen (CEA) were purchased from Abcam (USA). Prostate-specific antigen (PSA), bovine serum albumin (BSA) and serumfrom normal human male AB plasma were obtained fromSigma-Aldrich (USA). Hepatitis B surface antigen (HBsAg) wasacquired from Fitzgerald Industries International Inc. (USA).PB NPs were typically prepared according to the published lit-erature.19 Unless otherwise stated, all other chemicals were ofanalytical grade and used as received.

Preparation of antibody-conjugated iron oxide NPs

The polyclonal rabbit anti-human PSA antibody was covalentlyconjugated to carboxyl-functionalized iron oxide NPs throughthe typical carbodiimide method. Typically, 1.0 mg iron oxideNPs were dispersed in 2.0 mL deionized water with ultrasoni-cation. An aqueous mixture (25.0 μL) of N-hydroxysulfosuccini-mide (Sulfo-NHS) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC·HCl) with the same concen-tration of 25.0 mg mL−1 was added to the nanoparticle dis-persion, followed by reactions at room temperature for 30 minunder gentle stirring. 80.0 μg polyclonal rabbit anti-humanPSA antibody was then added into the above nanoparticle dis-persion, followed by reactions at room temperature for 2.0 hunder gentle stirring. The nanoparticle dispersion was centri-fuged at 11 000 rpm for 10.0 minutes at 4.0 °C to collect theantibody-conjugated iron oxide NPs, which were then washed3 times with PBS (pH = 7.4, 0.01 M). The antibody-conjugatediron oxide NPs were finally dispersed in 2.0 mL PBS (pH = 7.4,0.01 M) containing 0.2% BSA. The nanoparticle dispersionswere stored at 4.0 °C before use.

Fig. 1 Schematic illustration of the colorimetric immunoassay using theiron oxide-to-PB NP colorimetric conversion strategy.

Scheme 1 Schematic illustration of the iron oxide-to-PB NP conversionprocess.

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Procedures of the colorimetric immunoassay

A 100 μL monoclonal mouse anti-human PSA antibody solu-tion (30.0 μg mL−1) was added into each PCR tube (200 μL)and incubated for 12.0 h at 4.0 °C. A 200 μL blocking buffercontaining 5.0% BSA was then used to block the tubes for2.0 h at 37.5 °C, followed by incubation with different concen-trations of standard PSA solutions containing 5.0% BSA for2.0 h at 37.5 °C. After thorough washing, a 100 μL polyclonalanti-PSA antibody-conjugated iron oxide NP suspension(0.5 mg mL−1) was added into each tube for further incubationat 37.5 °C for 2.0 h. Finally, the PCR tubes were thoroughlywashed with PBS.

To transform iron oxide NPs captured in the sandwich-typeimmunosensing system into PB NPs, a 120 μL HCl solution(0.1 M) was added into each tube, followed by ultrasonicationfor 1.0 h at room temperature. A 30.0 μL potassium ferrocya-nide aqueous solution (90.0 mM) was then added into eachtube to produce PB NPs from the reaction between ferric ionsand ferrocyanide ions under acidic conditions. The immuno-sensing solutions were thoroughly mixed every 10 min, andwere finally used for UV-Vis spectroscopic characterization,Fourier transform infrared spectroscopic (FTIR) and trans-mission electron microscopic (TEM) characterization after thereaction for 1.0 h.

Characterization and instruments

UV-Vis spectrometry, FTIR and TEM were used to characterizethe immunosensing solutions before and after the iron oxide-to-PB NP conversion process. The UV-Vis spectra of theimmunosensing solutions were performed on a 96-well micro-plate using a SPECTROstar Nano Microplate Reader (BMGLABTECH). FTIR was performed on a Spectrum 100 FT-IRspectrometer (PerkinElmer, Inc.). Immunosensing solutions atthe PSA concentration of 64.0 ng mL−1 before and after theiron oxide-to-PB NP conversion process were dropped onWhatman® cellulose chromatography paper (Sigma-Aldrich),followed by air drying at room temperature for the FTIRmeasurement using the chromatography paper as the blank.TEM was carried out to observe the morphology of nano-particles in the immunosensing solutions using a JEOL3200FS cryo-transmission electron microscope. Immuno-sensing solutions at the PSA concentration of 64.0 ng mL−1

before and after the iron oxide-to-PB NP conversion processwere deposited on carbon-coated copper grids for TEMimaging. Additionally, photographs were taken with a CanonEOS 600D camera to record the color changes.

Colorimetric detection in human serum

Serum from normal humans was used for the real sampledetection to validate the reliability of the developed colori-metric immunoassay. 10.0 μL different concentrations of stan-dard PSA solutions were spiked into 1.0 mL human serumwhich was pre-diluted 3 folds with PBS to prepare the spikedserum samples with the final PSA concentrations of 4.0, 8.0and 16.0 ng mL−1, respectively. After thorough mixing, the

concentrations of PSA in the spiked serum samples weretested with the developed colorimetric immunoassay. Inaddition, to validate the analytical reliability of the developedcolorimetric immunoassay for detection of real human serumsamples, the conventional UV-Vis spectrometry was used tomeasure the PSA spiked in the human serum samples to calcu-late the spike recoveries.

Results and discussionCharacterization and confirmation of the nanoparticlecolorimetric conversion process

To confirm the generation of PB NPs in the immunosensingsolutions after the iron oxide-to-PB NP conversion process, col-orimetric, UV-Vis spectroscopic and Fourier transform infraredspectroscopic (FTIR) characterization were carried out. Fig. 2Ashows the photographs of the immunosensing solutionsbefore and after the nanoparticle conversion process. Noapparent color change was observed in the absence of thetarget PSA after the nanoparticle conversion process, indicat-ing the absence of PB NPs in the immunosensing solution,because no iron oxide NPs were captured in the immuno-sensing system without the target PSA. As expected, a clear

Fig. 2 Photographs (A) and UV-Vis spectra (B) of PB NP aqueous dis-persion and the immunosensing solutions at different PSA concen-trations before and after the nanoparticle conversion process. (C) FTIRof PB NPs and the immunosensing solution at the PSA concentration of64.0 ng mL−1 before and after the nanoparticle conversion process. Thereaction time of the nanoparticle conversion process was 1.0 h.

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color change from light brown to bright blue was observed inthe presence of 64.0 ng mL−1 PSA after the nanoparticle con-version process. The light brown color before the nanoparticleconversion process was attributed to the iron oxide NPs cap-tured in the immunosensing system. The bright blue colorafter the nanoparticle conversion process was consistent withthe typical color of PB NPs, revealing the generation of PB NPsin the immunosensing solution.

With the clear color change after the nanoparticle conver-sion process, a broad absorption peak was observed at 748 nmin the UV-Vis spectra of the immunosensing solution (64.0 ngper mL PSA) (Fig. 2B), while no absorption peak was exhibitedbefore the nanoparticle conversion process. The absorptionpeak corresponded well with that of PB NPs attributed to thecharge transfer transition between Fe(II) and Fe(III) in PBNPs,19,20 demonstrating the generation of PB in the immuno-sensing solution after the nanoparticle conversion process.The slight redshift of the absorption peak of PB in theimmunosensing solution might be attributed to the differentmatrix effect from the immunosensing solution. No noticeableabsorption peak was recorded both before and after the nano-particle conversion process in the absence of the target PSA,which indicated that no PB was generated in the absence ofthe target PSA. These results confirmed the successful ironoxide-to-PB conversion process in the presence of the targetPSA, providing the possibilities for colorimetric immuno-sensing of PSA.

Along with color changes and UV-Vis spectra, FTIR was uti-lized to confirm the generation of PB in the immunosensingsolution, as shown in Fig. 2C. As can be seen, an apparentstretching band was observed at 2085 cm−1 after the nano-particle conversion process, while no band was observedbefore the nanoparticle conversion process. Significantly, thestretching band corresponded well with that of PB NPs(2085 cm−1), demonstrating the successful iron oxide-to-PBconversion process in the presence of the target PSA. Thestretching band can be attributed to the CN stretching in theformed [FeII–CN–FeIII] structure in PB NPs.25,26

To further confirm the iron oxide-to-PB NP conversionprocess, TEM was used to study the morphological change ofnanoparticles in the immunosensing solutions. Fig. 3 shows

the TEM images of nanoparticles in the immunosensing solu-tions before and after the nanoparticle conversion process. Itcan be seen that before the conversion, a number of iron oxideNPs with uniformly spherical morphology with an average dia-meter of 40 nm were observed in the TEM image, which was ingood agreement with the product information from the manu-facturer (Ocean NanoTech LLC, USA). However, an obviouschange in morphology of the nanoparticles was observed afterthe nanoparticle conversion process. With the disappearanceof the spherical iron oxide NPs, nanoparticles with clear cubicmorphology of the size from 20 to 100 nm were observed inthe TEM image. The cubic morphology of the nanoparticleswas in good agreement with the well-known cubic morphologyof PB NPs.19,27,28 Spherical iron oxide NPs captured in thesandwich-type immunoassay system were first dissolved inacidic conditions under ultrasonication to release ferric ions(Fe3+), followed by the reaction between ferric ions and theadded ferrocyanide ions to produce PB NPs that had a typicalcubic morphology. The wide size distribution of PB NPs in theimmunosensing solution might be due to the absence ofsurface capping agents during the nucleation between ferricions and potassium ferrocyanide.28 These results further con-firmed the successful spherical iron oxide-to-cubic PB NP con-version process.

Colorimetric immunosensing using the nanoparticleconversion strategy

To study the feasibility of the iron oxide-to-PB NP conversionstrategy for colorimetric immunosensing, different concen-trations of standard PSA in bovine serum albumin (BSA) solu-tions were tested with the immunoassay method illustrated inFig. 1.

Fig. 4A shows that as the PSA concentration increased inthe range from 1.0 to 64.0 ng mL−1, a gradually deepening ten-dency from light yellow to bright blue in the color of theimmunosensing solutions was observed after the nanoparticleconversion process. As the PSA concentration increased, theamount of iron oxide NPs captured in the immunosensingsystem increased accordingly, thereby resulting in the concen-tration increase of PB NPs generated from the iron oxide-to-PBNP conversion process. It was reported that PB NPs showedhigh molar extinction coefficients comparable to that of goldnanorods in the near-infrared region (700–900 nm).19 There-fore, by employing PB NPs with high structural stability andsimple transformation as a colorimetric probe, the iron oxide-to-PB NP conversion process provides a new promising strategyfor cost-effective and easy-to-use colorimetric immunosensing.In addition, with the clear color change as the PSA concen-tration increased, the absorption peak of PB NPs at 748 nm inthe UV-Vis spectra also increased (Fig. 4B). Excitingly, it wasfound that the absorbance at 748 nm was proportional to thelogarithm of the PSA concentration in the range from 1.0 to64.0 ng mL−1, the common clinically relevant diagnosticlevel,29 with a correlation coefficient of 0.996 (Fig. 4C). Theresult demonstrated a good agreement between colorimetricimmunosensing and UV-Vis spectrometry.

Fig. 3 TEM images of nanoparticles in immunosensing solutions at thePSA concentration of 64.0 ng mL−1 before (A) and after (B) the nano-particle conversion process.

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It was also found that visible color difference between1.0 ng mL−1 PSA and the control can be distinguished by thenaked eye, as shown in Fig. 4A, suggesting high sensitivity ofthe colorimetric immunosensing method without the aid ofany nanoparticle stabilizing ligands and signal amplificationprocesses. The distinguishable color difference between1.0 ng mL−1 PSA and the control was further confirmed by theUV-Vis spectra as shown in Fig. 4B. Hence, PSA could bequickly detected at a concentration as low as 1.0 ng mL−1 withthe naked eye. Although this concentration is higher than thatof some electrochemical and fluorescence methods,30,31 it ismuch lower (∼80 folds) than that of the reported Au NP-basedcolorimetric PSA assay (80 ng mL−1),32 indicating the high-sensitivity of our method. This limit of detection (LOD) is alsocomparable to the conventional ELISA method (LOD: 1.0 ngmL−1) and commercial PSA ELISA kits (LOD: 1.0 ng mL−1,Biocell Biotechnol. Co., Ltd, Zhengzhou, China) using spec-trometers as reported in the published literature.33 Further-more, it is worth noting that the colorimetric immunoassaycan meet the requirement of clinical diagnostics because thethreshold concentration of PSA in human serum in prostatecancer diagnostics is 4.0 ng mL−1.33

Specificity of the colorimetric immunosensing strategy

To evaluate the specificity of the colorimetric immunosensingstrategy, some other common interfering substances in serumwith 10-fold higher concentrations than PSA (16.0 ng mL−1)including carcinoembryonic antigen (CEA), ImmunoglobulinG (IgG), and hepatitis B surface antigen (HBsAg) were testedwith the colorimetric immunoassay. It should be noted that4.0 ng mL−1 was used as the lowest PSA concentration forspecificity study because of its clinical diagnostic significanceas a lower threshold diagnostic concentration.33 Normalhuman serum was also used as an interfering substance. Asshown in Fig. 5A, only the immunosensing solution obtainedfrom the target PSA (4.0 and 16.0 ng mL−1) exhibited a clearcolor change to blue after the nanoparticle conversion process.In addition, the UV-Vis spectra (Fig. 5B) show that only theimmunosensing solution obtained from the target PSA hadobvious absorption at 748 nm, while other interfering sub-stances exhibited less than 4.7% absorbance in comparisonwith PSA (relative to blank). These results confirmed high anti-interference capability of the colorimetric immunosensingstrategy for PSA detection in the presence of high concen-trations of interfering substances.

Colorimetric immunosensing in human serum samples

To validate the analytical accuracy of the developed colori-metric immunosensing method for detection of real samples,serum samples from normal human were spiked with differentconcentrations of standard PSA for the colorimetric determi-nation. As an important lower threshold concentration for

Fig. 4 Photographs (A) and UV-Vis spectra (B) of the immunosensingsolutions at different PSA concentrations (5% BSA) before and after thenanoparticle conversion process. (C) Calibration plot of absorbance at748 nm in UV-Vis spectra vs. logarithm of the PSA concentration. Errorbars indicate standard deviations (n = 3).

Fig. 5 Specificity study. Photographs (A) and absorbance at 748 nm (B)of the immunosensing solutions obtained from the target PSA anddifferent interfering substances before and after the nanoparticle con-version process. Serum from normal human was pre-diluted 3 folds withPBS. Error bars indicate standard deviations (n = 3).

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clinical prostate cancer diagnostics,33 4.0 ng mL−1 was hereinselected as the lowest spiked concentration for the validationstudy.

A distinct difference in the color of the immunosensingsolutions was observed by the naked eye as shown in Table 1.As the concentration of PSA spiked in the serum increased, adeepening trend of the color to blue was exhibited, which wasconsistent with Fig. 4A. Additionally, in order to confirm theresults from color changes, conventional UV-Vis spectrometrywas further used to detect PSA in the spiked human serumsamples. The detection results obtained from the UV-Vis spec-troscopic measurement corresponded well with that of thecolor changes. The analytical recoveries were in the range from91.3–93.1%, which were within the acceptable criteria for bio-analytical method validation.34

Conclusions

In conclusion, a novel iron oxide-to-PB NP colorimetric conver-sion strategy has been developed for cost-effective and sensi-tive colorimetric assays. We have demonstrated its applicationin the sensitive detection of cancer biomarkers through a newcolorimetric immunoassay, during which spherical iron oxideNPs captured in a sandwich-type immunosensing system aretransformed into cubic PB NPs which exhibit highly visibleblue color and high extinction coefficients. Without the aid ofany complex nanoparticle stabilizing ligands and signal ampli-fication processes which are generally required for traditionalAu NP-based colorimetric assays, PSA as a model analyte canbe detected at a low concentration of 1.0 ng mL−1 by our newcolorimetric method. In comparison with the reported Au NP-based colorimetric assay,32 our method is much more sensi-tive, with the LOD ∼80 folds lower than that of the Au NP-based colorimetric assay. Additionally, the cost of our methodis much lower than the Au NP-based colorimetric method. Weestimated that the unit price of Au nanoparticles ($ mg−1) is∼48 000 folds more expensive than PB and iron oxide NPs (see

Table S1 in the ESI† for comparison details). These uniqueadvantages of the colorimetric immunosensing strategy makeit particularly promising for development of sensitive, cost-effective and convenient colorimetric POC bioassays for abroad range of bioanalytical and environmental applications,particularly in resource-poor settings. This conversion strategyalso provides potential for developing other types of colori-metric methods based on PB NPs. But it should be noted thatthere are still several limitations of this new method for POCapplication, such as the relatively long and complicated nano-particle transformation process. However, these limitationscan be addressed to enhance the potential for POC detectionby further optimizing the reaction conditions and simplifyingthe transformation procedure (e.g. carrying out the transform-ation reaction during ultrasonication). In addition, the inte-gration of the new method on a microfluidic paper-baseddevice using a colorimetric readout card35 or some softwarelike ImageJ36 for quantitative analysis will provide a newopportunity to achieve simpler, faster, more sensitive, moreobjective and user-friendly POC detection. This work is under-way in our group.

Acknowledgements

We would like to acknowledge the financial support from NIH/NIGMS (SC2GM105584) and NIH/NIAID (R21AI107415). Finan-cial support from NIH RCMI for the BBRC Pilot grant, UTEPfor the IDR and MRAP programs, University of Texas (UT)System for the STARS award, is also greatly acknowledged. Weare grateful to the support from Dr Chuan Xiao’s Group atUTEP for the spectroscopic measurement and Dr Bernal’sgroup for TEM images.

Notes and references

1 J. S. Sun, Y. L. Xianyu and X. Y. Jiang, Chem. Soc. Rev.,2014, 43, 6239–6253.

2 S. T. Sanjay, G. L. Fu, M. Dou, F. Xu, R. T. Liu, H. Qi andX. Li, Analyst, 2015, 140, 7062–7081.

3 M. Dou, S. T. Sanjay, M. Benhabib, F. Xu and X. Li, Talanta,2015, 145, 43–54.

4 P. Zuo, X. Li, D. C. Dominguez and B. C. Ye, Lab Chip,2013, 13, 3921–3928.

5 I. Al-Ogaidi, H. L. Gou, Z. P. Aguilar, S. W. Guo,A. K. Melconian, A. K. A. Al-Kazaz, F. K. Meng andN. Q. Wu, Chem. Commun., 2014, 50, 1344–1346.

6 Y. X. Li, M. Hong, Y. Q. Lin, Q. Bin, Z. Y. Lin, Z. W. Cai andG. N. Chen, Chem. Commun., 2012, 48, 6562–6564.

7 C. Chun, J. Joo, D. Kwon, C. S. Kim, H. J. Cha, M. S. Chungand S. Jeon, Chem. Commun., 2011, 47, 11047–11049.

8 X. Li, Z. Nie, C. Cheng, A. Goodale and G. Whitesides, inProc. Micro Total Analysis Systems, 2010, vol. 14,pp. 1487–1489.

Table 1 Detection of PSA spiked in human serum samples by the col-orimetric immunoassay (n = 4)

Serumsamplenumber

Standard PSAconcentration(ng mL−1)

Colorchanges

UV-Visdetectionresult(ng mL−1)

Recovery(%)

Control 0

No. 1 4.0 3.65 ± 0.20 91.3 ± 5.0

No. 2 8.0 7.32 ± 0.36 91.6 ± 4.6

No. 3 16.0 14.9 ± 0.90 93.1 ± 5.7

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9 W. S. Qu, Y. Y. Liu, D. B. Liu, Z. Wang and X. Y. Jiang,Angew. Chem., Int. Ed., 2011, 50, 3442–3445.

10 F. Xia, X. L. Zuo, R. Q. Yang, Y. Xiao, D. Kang, A. Vallee-Belisle, X. Gong, J. D. Yuen, B. B. Y. Hsu, A. J. Heeger andK. W. Plaxco, Proc. Natl. Acad. Sci. U. S. A., 2010, 107,10837–10841.

11 Z. Z. Sun, N. Zhang, Y. M. Si, S. Li, J. W. Wen, X. B. Zhuand H. Wang, Chem. Commun., 2014, 50, 9196–9199.

12 D. B. Liu, Z. Wang and X. Y. Jiang, Nanoscale, 2011, 3,1421–1433.

13 Y. J. Liu, L. Q. Zhang, W. Wei, H. Y. Zhao, Z. X. Zhou,Y. J. Zhang and S. Q. Liu, Analyst, 2015, 140, 3989–3995.

14 C. Chen, M. Luo, T. Ye, N. Li, X. Ji and Z. He, Analyst, 2015,140, 4515–4520.

15 P. Liu, X. H. Yang, S. Sun, Q. Wang, K. M. Wang, J. Huang,J. B. Liu and L. L. He, Anal. Chem., 2013, 85, 7689–7695.

16 L. H. Guo, Y. Xu, A. R. Ferhan, G. N. Chen and D. H. Kim,J. Am. Chem. Soc., 2013, 135, 12338–12345.

17 M. S. Verma, J. L. Rogowski, L. Jones and F. X. Gu, Bio-technol. Adv., 2015, 33, 666–680.

18 K. Saha, S. S. Agasti, C. Kim, X. N. Li and V. M. Rotello,Chem. Rev., 2012, 112, 2739–2779.

19 G. L. Fu, W. Liu, S. S. Feng and X. L. Yue, Chem. Commun.,2012, 48, 11567–11569.

20 G. L. Fu, W. Liu, Y. Y. Li, Y. S. Jin, L. D. Jiang, X. L. Liang,S. S. Feng and Z. F. Dai, Bioconjugate Chem., 2014, 25,1655–1663.

21 M. Espinoza-Castaneda, A. de la Escosura-Muniz,A. Chamorro, C. de Torres and A. Merkoci, Biosens. Bio-electron., 2015, 67, 107–114.

22 W. M. Zhang, D. Ma and J. X. Du, Talanta, 2014, 120, 362–367.

23 A. Teepoo, P. Chumsaeng, S. Jongjinakool, K. Chantu andW. Nolykad, J. Appl. Sci., 2012, 12, 568–574.

24 B. Zargar and A. Hatamie, Anal. Methods, 2014, 6, 5951–5956.

25 X. Q. Zhang, S. W. Gong, Y. Zhang, T. Yang, C. Y. Wang andN. Gu, J. Mater. Chem., 2010, 20, 5110–5116.

26 M. Shokouhimehr, E. S. Soehnlen, A. Khitrin, S. Basu andS. P. D. Huang, Inorg. Chem. Commun., 2010, 13, 58–61.

27 M. Hu, S. Furukawa, R. Ohtani, H. Sukegawa, Y. Nemoto,J. Reboul, S. Kitagawa and Y. Yamauchi, Angew. Chem., Int.Ed., 2012, 51, 984–988.

28 M. Shokouhimehr, E. S. Soehnlen, J. H. Hao, M. Griswold,C. Flask, X. D. Fan, J. P. Basilion, S. Basu andS. P. D. Huang, J. Mater. Chem., 2010, 20, 5251–5259.

29 A. I. Barbosa, A. P. Castanheira, A. D. Edwards andN. M. Reisa, Lab Chip, 2014, 14, 2918–2928.

30 X. Q. Chen, G. B. Zhou, P. Song, J. J. Wang, J. M. Gao,J. X. Lu, C. H. Fan and X. L. Zuo, Anal. Chem., 2014, 86,7337–7342.

31 J. Liu, C. Y. Lu, H. Zhou, J. J. Xu, Z. H. Wang andH. Y. Chen, Chem. Commun., 2013, 49, 6602–6604.

32 A. Drew, Honors Theses, 2015, Paper 113.33 Z. Q. Gao, L. Hou, M. D. Xu and D. P. Tang, Sci. Rep., 2014,

4, 3966.34 S. Y. Zhou, W. Zheng, Z. Chen, D. T. Tu, Y. S. Liu, E. Ma,

R. F. Li, H. M. Zhu, M. D. Huang and X. Y. Chen, Angew.Chem., Int. Ed., 2014, 53, 12498–12502.

35 J. Hu, S. Q. Wang, L. Wang, F. Li, B. Pingguan-Murphy,T. J. Lu and F. Xu, Biosens. Bioelectron., 2014, 54, 585–597.

36 M. Dou, D. C. Dominguez, X. Li, J. Sanchez and G. Scott,Anal. Chem., 2014, 86, 7978–7986.

Analyst Paper

This journal is © The Royal Society of Chemistry 2016 Analyst, 2016, 141, 3883–3889 | 3889

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