Analytical Biochemistry 399 (2010) 72–77

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Analytical Biochemistry

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Chemiluminescent immunoassay of thyroxine enhancedby microchip electrophoresis

Yong Huang a, Shulin Zhao a,b,*, Ming Shi a, Yi-Ming Liu b,*

a Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering,Guangxi Normal University, Guilin 541004, Chinab Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 September 2009Received in revised form 25 November 2009Accepted 30 November 2009Available online 2 December 2009

Keywords:ThyroxineHomogeneous competitive immunoassayMicrochip electrophoresisChemiluminescence detectionThyroid gland function diagnosis

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.11.036

* Corresponding authors. Fax: +1 601 979 3674.E-mail addresses: zhaoshulin001@163.com (S.

(Y. -M. Liu).1 Abbreviations used: T4, thyroxine; RIA, radioimmun

fluorescence immunoassay; Ab, antibody; MCE, mcapillary electrophoresis; IgG, immunoglobulin G; LIFCL, chemiluminescence; MCE–CL, MCE with CL dealbumin; IAP, immunosuppressive acidic protein; HHRP–T4, HRP-labeled T4; PIP, para-iodophenol; H2O2,sodium hydrogen carbonate; PMT, photomultiplier tuane; S, sample reservoir; SW, sample waste reservoir;waste reservoir; R, oxidizer solution reservoir; HPchromatography; S/N, signal/noise ratio; RSD, relative

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophore-sis separation has been developed. The method deployed the competitive immunoreaction of T4 andhorseradish peroxidase (HRP)-labeled T4 (HRP–T4) with anti-T4 mouse monoclonal antibody (Ab).HRP–T4 and the HRP–T4–Ab complex were separated and quantified by using microchip electrophoresis(MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means ofHPR-catalyzed luminol–H2O2 reaction. Due to the effective MCE separation, the CL analytical signal wasless prone to sample matrix interference. Under the selected assay conditions, the MCE separation wasaccomplished within 60 s. The linear range for T4 was 5–250 nM with a detection limit of 2.2 nM (sig-nal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in humanserum samples. It was demonstrated that the current MCE–CL-enhanced competitive immunoassaywas quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist inthe diagnosis of thyroid gland functions.

� 2009 Elsevier Inc. All rights reserved.

1 0 0

Thyroxine (T4, 3,5,3 ,5 -tetraiodo-L-thyronine) is the primary ac-tive hormone synthesized within the follicular cells of the thyroidgland [1]. It affects metabolic activity in many tissues, leading to in-creased consumption of oxygen and stimulation of mitochondrialrespiration. Measurement of the serum T4 level is commonly usedfor diagnosis of thyroid gland diseases such as hypothyroid, hyper-thyroid, thyroidectomy, and thyroiditis. Assays currently used inclinical practice include radioimmunoassay (RIA), chemiluminescentenzyme immunoassay, and time-resolved fluorescence immunoas-say (TRFIA) [2]. All of them involve immunoreactions of T4 withanti-T4 antibodies (Abs). However, due to differences in reagentspecificity, the concentration of free T4 in a given specimendetermined with assays from different manufacturers can vary. In

ll rights reserved.

Zhao), yiming.liu@jsums.edu

oassay; TRFIA, time-resolvedicrochip electrophoresis; CE,, laser-induced fluorescence;

tection; HSA, human serumRP, horseradish peroxidase;hydrogen peroxide; NaHCO3,

be; PDMS, polydimethylsilox-B, buffer reservoir; BW, bufferLC, high-performance liquidstandard deviation.

addition, heterophile Ab interference with T4 quantification thatcaused clinical confusions has been reported [3,4]. To improve thereliability of assay results, mass spectrometry-based analytical pro-tocols have been developed recently [5,6].

Microchip electrophoresis (MCE), regarded as a miniaturizedversion of capillary electrophoresis (CE), has become a very attrac-tive separation technique [7]. It offers many advantages, such asminiaturized apparatus, extremely small sample size, high separa-tion speed and efficiency, short analysis time, and ease of integra-tion and automatization, that make it unequally suitable forbiological and clinical analysis. The technique has been success-fully applied to separation of chemical species of biomedical inter-est, including amino acids [8,9], biogenic amines [10], proteins[11,12], and DNA [13,14]. Immunoassay is known as one of themost important and widely used analytical techniques in clinicaldiagnoses and biochemical studies. Performing immunoassays bymeans of microfluidic devices is currently gaining research inter-est. Incorporation of a microfluidic system in an immunoassay sig-nificantly simplifies the procedure and offers advantages, includinghigh separation and reaction efficiency, shortened assay time, andlower sample, reagent, and energy consumption. Over the pastdecade, MCE-enhanced immunoassay of cortisol [15], theophylline[16], 2,4,6-thrinitrotoluene [17], rat immunoglobulin G (IgG) [18],insulin [19,20], and inflammatory cytokines [21] has beenreported. However, in most of these works, laser-induced fluores-cence (LIF) detection was deployed for detecting the separated

Chemiluminescent immunoassay of thyroxine / Y. Huang et al. / Anal. Biochem. 399 (2010) 72–77 73

species. LIF detection requires relatively large and expensiveapparatus systems. Chemiluminescence (CL) detection offersadvantages, such as simplicity in instrumentation, high sensitivity,and wide linear range, and is particularly suitable for integrationon microfluidic devices. Methods based on MCE with CL detection(MCE–CL) have been developed for analysis of amino acids [22],biogenic amines [23], peptides [24], proteins [25], and the like.MCE–CL-enhanced immunoassay for the determination of rat IgG[26], as well as human serum albumin (HSA) and immunosuppres-sive acidic protein (IAP) [27], has also been reported.

In this work, we report on the development of an MCE–CL-en-hanced homogeneous immunoassay of T4. It is well known thathorseradish peroxidase (HRP) catalyzes luminol–H2O2 CL reactionand greatly enhances the CL emission. Therefore, HRP-labeled T4(HRP–T4) was selected as the competing reagent of T4 in the sam-ple for anti-T4 Ab. Both HRP–T4 and the HRP–T4–Ab complex weresensitively detected by CL after MCE separation. The use of MCEseparation might also improve the assay selectivity by isolatingHRP–T4 from other potentially chemiluminescent species. TheMCE–CL-enhanced competitive immunoassay was preliminarilyvalidated by quantifying T4 in serum samples taken from patientssuffering from various thyroid diseases.

Materials and methods

Chemicals and reagents

Luminol was purchased from Fluka (Buchs, Switzerland). Para-iodophenol (PIP), hydrogen peroxide (H2O2), Na3PO4, and sodiumhydrogen carbonate (NaHCO3) were obtained from Taopu Chemi-cals (Shanghai, China). A T4 assay kit (lot 08280), which consistedof T4 standards containing 0, 5, 15, 50, 150, and 500 lg/L T4 inhuman serum, T4 controls (containing low and high concentrationsof T4 in human serum), T4 enzyme conjugate (HRP–T4), and anti-T4 mouse monoclonal Ab, was obtained from Diagnostics SystemsLaboratories (Webster, TX, USA). All other chemicals were of ana-lytical grade. Ultrapure water (18.2 MX) prepared from double-distilled water with Millipore Simplicity was used throughoutthe work. The electrophoresis buffer was 10 mM Na3PO4 (pH10.2) containing 1.2 mM luminol and 0.005% (w/v) Brij 35, andthe oxidizer solution was 35 mM NaHCO3 (pH 8.6) containing100 mM H2O2 and 1.2 mM PIP. All solutions were filtered through0.22-lm membrane filters prior to use.

MCE–CL system

The lab-built MCE–CL system was described previously [28].Briefly, the microchip assembly was mounted on the x–y transla-

Fig. 1. Layout and dimension of the glass/PDMS hybrid microchip. S, sample reservoir; B,solution reservoir.

tional stage of an inverted microscope (Olympus CKX41) that alsoserved as a platform for CL detection. CL signal was collected bymeans of a microscope objective and detected by a photomultipliertube (PMT, Hamamatsu R105). Signals from the PMT wererecorded and processed with a computer using a ChromatographyData System (Zhejiang University Star Information Technology,Hangzhou, China). A multiterminal high-voltage power supply,variable in the range of 0–8000 V (Shandong Normal University,Jinan, China), was used for sample injection and MCE separation.The inverted microscope was placed in a black box.

The fabrication of the glass/polydimethylsiloxane (PDMS)microchip was described previously [23,24]. Its schematic layoutis illustrated in Fig. 1. The width of all microchannels except theoxidizer introduction channel (250 lm) was 70 lm, the depth ofall microchannels was 25 lm, and the length of double T was60 lm. All reservoirs were 4.0 mm in diameter and 1.5 mm deep.The channel between the sample reservoir (S) and the samplewaste reservoir (SW) was used for sampling, the channel betweenthe buffer reservoir (B) and the buffer waste reservoir (BW) wasused for separation, and the channel between the oxidizer solutionreservoir (R) and BW was used for oxidizer introduction.

Pretreatment of human serum samples

Human serum samples were kindly provided by the No. 5 Peo-ple’s Hospital (Guilin, China). To 500 ll of a serum sample, 0.5 mlof a sulfosalicylic acid solution (5 mg/ml) was added. The mixturewas votexed and left to stand for 5 min at room temperature torelease free T4 from protein-conjugated T4 [29,30]. The solutionwas centrifuged (12,000g for 10 min). The supernatant was trans-ferred into a centrifuge tube and diluted to 2 ml. The pH of thesolution was adjusted to approximately 7.4. The obtained solutionwas kept at –20 �C before analysis.

Immunoreaction

To carry out the immunoreaction, 20 ll of T4 standards or ser-um samples was mixed with 20 ll of 6.0 � 10–7 M HRP–T4 and20 ll of 4.0 � 10–7 M mouse anti-T4 monoclonal Ab in a 0.5-mlmicrocentrifuge tube. The solution was incubated for 15 min at37 �C before an MCE–CL run.

MCE–CL operation

All of the microchannels on the microchip were sequentiallywashed with 0.1 M NaOH, water, and electrophoresis buffer for1 min each before each run. The microchannels were filled withelectrophoresis. The sample was transferred into S. SW, B, and

buffer reservoir; SW, sample waste reservoir; BW, buffer waste reservoir; R, oxidizer

74 Chemiluminescent immunoassay of thyroxine / Y. Huang et al. / Anal. Biochem. 399 (2010) 72–77

BW were filled with electrophoresis buffer, and R was filled withthe oxidizer solution (H2O2). Platinum wires as electrodes were in-serted into these reservoirs. Sample injection was performed byapplying 800 V to S for 15 s with SW grounded, whereas B wasset at 250 V, BW was set at 500 V, and R was left floating. To carryour MCE separation, 2800 V was applied to B and 2500 V was ap-plied to both S and SW with BW grounded. At the same time,550 V was applied to R. The analyte was transported into the sep-aration channel toward BW and then was mixed with the oxidizersolution at the junction of the oxidizer introduction channel andthe separation channel, producing CL emission that was collectedthrough a microscope objective and then detected by a PMT.

Fig. 3. pH Effects of the oxidizer solution on CL intensity. Electrophoresis buffer was10 mM phosphate buffer (pH 10.2) containing 0.005% (w/v) Brij 35. The oxidizersolution was 35 mM NaHCO3 containing 100 mM H2O2 and 1.2 mM PIP at varyingpH values. The concentration of HRP–T4 was 3.0 � 10–7 M.

Results and discussion

Optimization of MCE–CL conditions

Effective separation of Ag* and Ag*–Ab is a key step for successin all competitive immunoassays. In this work, MCE was used toaccomplish the separation. However, it was found that adsorptionof proteins by the glass channel surface significantly deterioratedthe separation of HRP–T4 from the HRP–T4–Ab complex. To over-come this difficulty, a surface-active agent, Brij 35, was added tothe electrophoresis buffer. The effect of Brij 35 concentration in arange of 0.002–0.006% (w/v) was investigated. The results areshown in Fig. 2. It can be seen that the resolution of HRP–T4 andthe HRP–T4–Ab complex increased with increasing Brij 35 concen-trations. However, at high Brij 35 concentrations, the migrationtimes of both HRP–T4 and HRP–T4–Ab were increased signifi-cantly. A concentration of 0.005% Brij 35 was selected for anacceptable resolution and a favorable separation time. Other MCEseparation conditions, such as buffer pH and separation voltage,were also studied. The pH of the electrophoresis buffer was testedin a range from 9.0 to 10.5. HRP–T4 and HRP–T4–Ab were bestresolved at pH 10.2. The separation voltage directly affected themigration rate and peak shape of analytes. After a careful study,2800 V was selected to achieve a good separation within a shortseparation time (<60 s).

As in all conventional chemiluminescent immunoassays,enhancing the sensitivity of CL detection improves assay sensitiv-

Fig. 2. Effects of Brij 35 concentration on resolving HRP–T4 and HRP–T4–Abcomplex. Electrophoresis buffer was 10 mM phosphate buffer (pH 10.2) containingBrij 35 at varying concentrations. The oxidizer solution was 35 mM NaHCO3

(pH 8.6) containing 100 mM H2O2 and 1.2 mM PIP.

ity. To maximize the sensitivity of CL detection following MCE sep-aration, the conditions such as concentrations of luminol, H2O2,PIP, and the pH of the oxidizer solution were optimized. In thisMCE–CL system, HRP–T4 was used as the competitive reagentand, therefore, was used as the test compound in the optimizationstudies. As shown in Fig. 3, the CL intensity from HRP–T4 increasedrapidly with increases in the pH of the oxidizer solution, reachingthe maximum at pH 8.6 before decreasing dramatically. It wasfound that the concentrations of H2O2 and luminol also signifi-cantly affected the CL intensity. The optimal concentrations were1.2 mM for luminol and 100 mM for H2O2. To further improvethe detection sensitivity, PIP was added as a CL enhancer [31].The effect of PIP concentration on the CL intensity is illustratedin Fig. 4. The optimum PIP concentration was 1.2 mM. In the pres-ence of PIP, CL intensity increased approximately eight times.

Fig. 4. Effects of PIP concentration on CL intensity. The oxidizer solution was35 mM NaHCO3 (pH 8.6) containing 100 mM H2O2 and PIP at varying concentra-tions. Other conditions were as in Fig. 3.

Fig. 6. Electropherograms from separating the competitive immunoreaction solu-tions: (a) 6.0 � 10–7 M HRP–T4 solution; (b) mixture of 6.0 � 10–7 M HRP–T4 and3.0 � 10–7 M anti-T4 antibody; (c) mixture of 8.0 � 10–8 M T4, 6.0 � 10–7 M HRP–T4,and 4.0 � 10–7 M anti-T4 antibody. MCE–CL conditions were as in Fig. 5.

Chemiluminescent immunoassay of thyroxine / Y. Huang et al. / Anal. Biochem. 399 (2010) 72–77 75

Competitive immunoassay for T4 by MCE–CL

In this work, a competitive format was adopted to determinefree T4. The format can be described as follows:

Agþ Ag� þ Ab ¼ Agþ Ag� þ Ag—Abþ Ag�—Ab;

where Ab is anti-T4 Ab added at a limited amount, Ag* is HRP–T4added at a fixed amount, and Ag is free T4. HRP–T4 competes withfree T4 in the sample for binding to a limited amount of anti-T4 Ab.The concentration of T4 in the sample is directly proportional tothat of free HRP–T4 but inversely proportional to that of the HRP–T4–Ab complex. Thus, the concentration of T4 in the sample canbe determined by measuring the CL signal from HRP–T4 after beingseparated from the HRP–T4–Ab complex.

The incubation time for the immunoreaction was studied. TheHRP–T4 and Ab mixture was incubated for 4–20 min and thenwas injected into the MCE–CL system to determine the optimalincubation time when the maximum binding yield was obtained.The results are summarized in Fig. 5. As can be seen, the CL signalfrom the HRP–T4–Ab complex increased rapidly with increasingincubation times until 15 min at 37 �C and then remained constantuntil 20 min. These results indicate that the binding of HRP–T4 toanti-T4 Ab reached an equilibrium at 15 min. Therefore, an incuba-tion time of 15 min was selected for further experiments.

Fig. 6 shows typical electropherograms of the competitiveimmunoassay of T4. Trace a was obtained from a solution contain-ing HRP–T4 only. Trace b was obtained from a solution containingHRP–T4 and anti-T4 Ab. A new peak at a longer migration time thatwas from the HRP–T4–Ab complex was observed. Trace c was ob-tained from a solution containing T4, HRP–T4, and anti-T4 Ab.Compared with trace b, the CL signal (peak height) from HRP–T4increased and that from the HRP–T4–Ab complex decreased. Theseresults indicate the competitive binding of T4 to the Ab. It is worthnoting that the HRP–T4–Ab complex was well separated from freeHRP–T4, making the current assay very selective and useful forclinical sample analysis.

An advantage of the current assay is the short analysis time ofapproximately 20 min. Because the Ag + Ag* + Ab incubation iscarried out in a homogeneous solution in this assay, the binding

Fig. 5. Incubation time and the immunoreaction yield. A mixture prepared from20 ll of 6.0 � 10–7 M HRP–T4 and 20 ll of 4.0 � 10–7 M mouse anti-T4 monoclonalAb was incubated at 37 �C and analyzed at different time intervals from 4 to 20 min.Electrophoresis buffer was 10 mM phosphate buffer (pH 10.2) containing 0.055%(w/v) Brij 35. The oxidizer solution was 35 mM NaHCO3 (pH 8.6) containing100 mM H2O2 and 1.2 mM PIP. See text for other conditions.

equilibrium can be reached within 15 min. However, an incubationtime of 45 min or longer is normally required in the widely usedheterogeneous immunoassays based on Ab-coated microplates[2] or magnetic particles [32] due to the limited interaction amongthe analyte, the reagents, and the immobilized Ab. Furthermore,the MCE separation of Ag*–Ab from Ag* in this assay is very quick(<1 min), representing a distinct advantage of MCE over other sep-aration techniques such as high-performance liquid chromatogra-phy (HPLC) and CE. In a study reported previously, CE wasdeployed in a homogeneous immunoassay of T4 and the CE sepa-ration time used was 20 min [33].

Fig. 7. Electropherograms from analyzing three human serum samples: (a) from ahealthy subject; (b) from a thyroidectomy patient; (c) from a hypothyroid patient.MCE–CL conditions were as in Fig. 5.

Table 1Analytical results of T4 content in healthy and patient serum samples.

Thyroid condition T4 found (10–8 M) RSD (%, n = 7) Added (10–8 M) Total found (10–8 M) Recovery (%)

Euthyroid 11.5 3.4 10.0 21.8 103Euthyroid 8.2 2.3 10.0 17.8 96.0Euthyroid 15.3 2.7 10.0 25.3 100Euthyroid 13.9 3.6 10.0 23.7 98.0Euthyroid 12.3 1.9 10.0 22.8 105

Hypothyroid 3.8 4.8 5.0 8.5 94.0Hypothyroid 4.2 3.1 5.0 9.1 98.0Hypothyroid 5.0 2.2 5.0 9.7 94.0

Hyperthyroid 20.1 3.7 20.0 39.0 94.5Hyperthyroid 18.8 3.5 20.0 38.1 96.5

Thyroidectomy 35.6 2.5 40.0 74.7 97.8

Goitre 14.6 2.0 20.0 33.8 96.0Goitre 13.5 3.9 20.0 33.8 102

Thyroiditis 9.4 2.4 10.0 19.1 97.0Thyroiditis 11.2 3.0 10.0 21.1 99.0

76 Chemiluminescent immunoassay of thyroxine / Y. Huang et al. / Anal. Biochem. 399 (2010) 72–77

Analytical figures of merit

The method was evaluated in terms of response linearity, limitof detection, and reproducibility. The CL signal (peak height) fromfree HRP–T4 was used for quantification of T4. Under the opti-mized conditions, seven standard T4 solutions at various concen-trations from 5 to 250 nM T4 were analyzed. Linear regressionanalysis of the results yielded the following equation:

H ¼ 6:898C þ 0:624 r2 ¼ 0:9927;

where H is the relative CL intensity (lV) from HRP–T4 and C is theconcentration of T4 (nM). The calibration curves showed excellentlinearity with a correlation coefficient of 0.993. Based on a signal/noise ratio (S/N) of 3, the detection limit for T4 was estimated tobe 2.2 nM. Assay reproducibility was investigated by analyzing astandard solution 11 times. The results showed that relative stan-dard deviations (RSDs) of peak heights and migration times were3.1% and 4.8%, respectively, for HRP–T4 and 3.2 and 4.1%, respec-tively, for HRP–T4–Ab.

Quantification of T4 in human serum

Human serum samples taken from 5 healthy volunteers (euthy-roid) and 10 patients suffering from different thyroid diseases wereanalyzed to demonstrate the feasibility of the current MCE–CL-en-hanced immunoassay. Electropherograms obtained from analyzingthree serum samples are shown in Fig. 7. As can be seen, HRP–T4peak heights varied from sample to sample, indicating thevariation of free T4 content in these samples. It is also worth notingthat the peak heights from HRP–T4 and the HRP–T4–Ab complexchanged accordingly; that is, when the HRP–T4 peak became high-er, the HRP–T4–Ab complex peak was lower. This was the expectedresult of a competitive immunoreaction. From our analysis, theserum level of T4 in healthy subjects was in the range from8.2 � 10–8 to 15.3 � 10–8 M. This result was consistent with thatreported in the literature (6.4–15.4 � 10–8 M) [34]. Serum T4results measured in this work for healthy subjects and thyroidpatients are shown in Table 1. Serum samples taken from patientshaving hypothyroid, hyperthyroid, thyroidectomy, goitre, and thy-roiditis were analyzed. The highest level of T4 was detected for thethyroidectomy patients at 3.56 � 10–7 M (n = 1), and the lowestlevel was detected for the hypothyroid group with an average of4.33 � 10–8 M (n = 3). To evaluate the assay reliability, recoveryof T4 from the serum sample matrix was determined. The resultsare also summarized in Table 1. Recovery was from 94.0% to

105.0% with RSDs less than 4.8% (n = 7) for all of the samplesanalyzed.

Concluding remarks

In this work, an MCE–CL-enhanced immunoassay for quantifyingT4 in serum samples has been developed. The immunoreaction in-volved was based on the competitive format, where free T4 com-peted with HRP-labeled T4 for binding anti-T4 Ab. After MCEseparation, both HRP–T4 and the HRP–T4–Ab complex were sensi-tively detected by measuring the CL emission from HRP-catalyzedluminol–H2O2 reaction. Due to the high emission efficiency of theCL reaction, the sensitivity of the assay was very high with a detec-tion limit of 2.2 nM T4 (S/N = 3). More important, separating HRP–T4 from other potentially chemiluminescent species by MCE madethe CL analytical signal much less prone to the influence from thesample matrix, thereby likely improving the assay selectivity. Thecurrent MCE–CL-enhanced immunoassay was preliminarily vali-dated by analyzing human serum samples taken from both healthyvolunteers and patients suffering from different thyroid diseases.The serum level of T4 in healthy subjects was 1.22 ± 0.27 � 10–7 M(n = 5), whereas the T4 level was found to be 3.56 � 10–7 M in thy-roidectomy patients (n = 1) and 4.33 � 10–8 M (n = 3) in hypothyroidpatients. As demonstrated in this work, combining commerciallyavailable immunoassay kits containing an HRP-labeled antigenand an antibody with an MCE–CL platform promises to developimmunoassays offering not only improved sensitivity and selectivitybut also advantages such as a simplified procedure, a shortened anal-ysis time, and very low consumption of expensive reagents for clin-ical analysis.

Acknowledgments

Financial support from the National Natural Science Founda-tions of China (NSFC, grants 20665002 and 20875019 to S.Z.) andthe U.S. National Institutes of Health (grant S06GM08047 toY-M.L.) is gratefully acknowledged.

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