Anal. Chem. 2004, 76, 2203-2209
Immunophenotyping of Acute Leukemia Using an Integrated Piezoelectric Immunosensor Array Hua Wang,† Hui Zeng,‡ Zhimin Liu,† Yunhui Yang,† Ting Deng ,† Guoli Shen,*,† and Ruqin Yu*,†
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China, and Xiangya Medical College, Central South University, Changsha 410078, P. R. China
Immunophenotyping evaluation is of particular importance for the clinical diagnosis, therapy, and prognosis of acute leukemia. In this paper, an integrated piezoelectric immunosensor array has been developed for the first time to detect the differentiated leukocyte antigens for immunophenotyping of acute leukemia. The probes (crystals) of the array were fabricated with plasma-polymerized n-butylamine film and nanometer-sized gold particles on which the Fab′-SH fragments obtained by the reduction of leukemic lineage-associated monoclonal antibodies (markers) were subsequently immobilized. Investigation results showed that the developed immunosensor array could rapidly identify normal cells from leukemic blasts and define the leukemic blasts within certain phenotypic groups (lineages) by only one analysis of the sample purified or unpurified. It permits the detection of unpurified leukocytes in the dynamic concentration range of 2 orders of magnitude (104-106 cells mL-1). Up to 17 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with 8 M urea. Moreover, the piezoelectric immunoassay system was applied to evaluate a number of practical specimens with immunophenotyping results in acceptable agreement with those clinically classified. The newly proposed multiparameter analysis technique provides a rapid, simple, and direct alternative tool for clinical immunophenotyping of acute leukemia. According to the predominant phenotypic cell types, acute leukemias are generally classified into acute lymphoid leukemia (ALL) with T-cell and B-cell lineages and acute myeloid leukemia (AML) (sometimes called acute nonlymphocytic leukemia or ANLL) with granulocyte and monocyte lineages. This phenotypic information is invaluable for clinical selection of the therapeutic strategy and assessment of prognosis of acute leukemia.1 Immunophenotyping, which utilizes antibodies (usually monoclonal) to recognize various differentiated antigens of leukocytes, is a vitally important means for defining certain phenotypic lineages or * Corresponding author. E-mail:
[email protected]. Fax: (+86) 731-8821818. † Hunan University. ‡ Central South University. (1) Hoffman, R.; Edward J.; Banz, J.; Shattil, S. J.; Furie, B.; Cohen, H. J.; Silberstein, L. E.; McGlave, P.; Strauss, M.; Benz, E. J. In Hematology: Basic Principles and Practice, 3rd ed.; Hoffman, R., et al., Eds.; Churchill Livingstone Press: New York, 2000; pp 1007-1008, 1075. 10.1021/ac035102x CCC: $27.50 Published on Web 03/19/2004
© 2004 American Chemical Society
subsets of acute leukemias, especially for evaluating morphologically atypical and undifferentiated or suspected hybrid leukemias.1-3 Immunophenotyping decision-making generally requires the determination of multiple lineage-associated parameters by use of combined monoclonal antibodies that have been grouped together in clusters of differentiation (CD), such as CD2 and CD7 for T-cell ALL, CD10 and CD19 for B-cell ALL, CD13 and CD33 for granulocyte AML, and CD14 exclusively for monocyte AML.1-3 The main immunophenotyping methods now in clinical use are, however, either low sensitivity and time-consuming, such as the immunofluorescence microscopy, or complicated with the need for expensive instrumentation and technical skills, i.e., the flow cytometry (FCM) that is applied to date in very few laboratories.2 Therefore, exploring an improved simple and rapid methodology for immunophenotyping of acute leukemia is of considerable interest. The quartz crystal microbalance (QCM) immunosensor, which offers some advantages, including high sensitivity, low cost, realtime output, and label- or radiation-free entities, has been the active subject of investigations of biomolecular interactions and clinical bioassays.4,5 The QCM immunosensor as a mass-sensitive transducer device exhibits extremely high detection sensitivity for biological macromolecules including proteins, microbes, and whole cells. For example, Muramatsu et al.6 first developed a piezoelectric cell immunosensor for the quantitative detection of Candida albicans in the concentration range of 106-5 × 108 cells mL-1. The development of a QCM immunosensor commonly requires immobilization of antibodies (or in some cases antigens) on the transducer surface. It is preferable that antibodies be immobilized with highly controlled orientation so as to maximize their antigen-binding efficiency and attain ultimate sensitivity and selectivity of immunoassay. There are two well-established procedures for achieving this purpose, one through utilizing protein A (or protein G) to specifically bind the Fc regions of the immunoglobulins7-9 and the other by use of antibody native thiol (2) Ba, D. N. Modern Immunological Techniques and Applications; Beijing Medical University Press: Beijing, 1998; pp 877-883. (3) Traweek, S. T. Am. J. Clin. Pathol. 1993, 99, 504-512. (4) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (5) O’Sullivan, C. K.; Vaughan, R.; Guilbault, G. G. Anal. Lett. 1999, 32, 23532377. (6) Muramatsu, H.; Kajiwara, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1986, 188, 257-261. (7) Foresgren, A.; Sjo ¨quist, J. J. J. Immunol. 1966, 97, 822-827. (8) Derek, A. P.; Martin, T. F.; James, N. M. Analyst 1994, 119, 2769-2776. (9) Saha, K.; Bender, F.; Gizeli, E. Anal. Chem. 2003, 75, 835-842.
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groups at the Fab′-SH fragment hinge.10-13 The latter immobilization protocol is thought to have an advantage over existing methods because antibodies are orientedly immobilized with both high antigen-binding constants and operational stability or adhesion.10,12 Nevertheless, the activities of the immobilized Fab′-SH fragments can be largely influenced by the surface properties of the transducer and the chemical linkage used.13 Popular as the direct immobilization of Fab′-SH fragments onto bulk gold surface is, it might inevitably cause a loss in the activity of the bound antibody fragments resulting from gold-induced denaturation of proteins.10,11 In the present work, we have developed an integrated QCM immunosensor array composed of four kinds of leukemic lineageassociated probes to explore the differentiated leukocyte antigens for immunophenotyping of acute leukemia. A new Fab′-SH fragment-based immobilization strategy for antibodies was proposed coupling with plasma-polymerized film (PPF) and nanometer-sized gold (abbreviated as nanogold) particles. The immunosensing probes were initially modified with n-butylamine PPF to generate amine groups on the surfaces available to capture nanogold particles from the solution, resulting in nanogold particlederivatized interfaces. Fab′-SH fragments, which were obtained by the reduction of leukocyte-specific monoclonal antibodies with 2-mercaptoethylamine (2-MEA),10,11 were subsequently immobilized on the nanogold particle-derivatized surface of the probes, each probe for one of the aforementioned leukemic lineages. The resulted four kinds of probes were inserted around a laboratorymade detection vessel, forming an integrated immunosensor array for immunophenotyping (schematically illustrated in Figure 1). Highlights are focused on the investigations of direct identification and quantitative analysis of leukemic samples in whole blood by the proposed QCM probe array that could analyze sample once with simultaneous output of multiple leukemic lineage-associated parameters. Moreover, a number of clinical specimens were evaluated by the QCM immunoassay system with analytical results compared to those given by the immunological enzyme-labeling assay (IEA) and the FCM clinically used. The attractive response performances of the proposed QCM immunosensing system and its potential merits for immunophenotyping are described in detail. EXPERIMENTAL SECTION Apparatus. A capacitively coupled radio frequency plasma system was purchased from Beijing Institute of Electronics (Beijing, China). The quartz crystal microbalances (AT-cut, 9 MHz, gold electrodes) were obtained from Chenxing Radio Equipment (Beijing, China), one side of which was sealed with an O-ring of silicone rubber covered by a plastic plate forming an air compartment isolated from aqueous solution. A laboratory-made transistor-transistor logic integrated circuit (TTL-IC) was designed to drive the crystals at their resonance frequencies, which were monitored by high-frequency counters (model FC 1250, Wellstar). The prepared QCM probes for immunophenotyping were integrated around a laboratory-made detection vessel containing an (10) Karyakin, A. A.; Presnova, G. V.; Rubtsova, M. Y.; Egorov, A. M. Anal. Chem. 2000, 72, 3805-3811 (11) O’Brien, J. C.; Jones, V. W.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 2000, 72, 703-710. (12) Ihalainen, P.; Peltonen, J. Langmuir 2002, 18, 4953-4962. (13) Lu, B.; Xie, J. M.; Lu, C. L.; Wu, C.; Wei, Y. Anal. Chem. 1995, 67, 83-87.
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Figure 1. Schematic diagrams of the QCM immunosensor array integrated into the detection vessel in cutaway view (a) and top view (b).
assay buffer solution gently agitated by a magnetic stirrer (model JB-2, Shanghai Analytical Instruments, Shanghai, China). Experimental temperatures were controlled by use of a thermostat (model CSS 501, Chongqing Experimental Equipments, Chongqing, China). Materials. Nanogold particle suspensions (15 nm in diameter) were purchased from Sino-America Biological Products (Beijing, China). The nanoparticle suspensions were further purified and concentrated by dialysis treatment resulting in concentration approximate to 1.25 × 1016 particles mL-1. N-Butylamine, ethylendiaminetetraacetic acid (EDTA), and urea were the products of Wuhan Organic Chemicals (Wuhan, China). 2-MEA was obtained from Sigma-Aldrich. Anti-leukocyte monoclonal antibodies CD2, CD7, CD10, CD19, CD13, CD33, and CD14 were obtained from Xiehe Research Institute of Hematopoietic Stem Cell (Tianjin, China). Acute leukemic and normal cell samples purified and unpurified in whole blood (∼1.0 × 107 cells mL-1) and clinical specimens were kindly provided by the Clinical Laboratory of Xiangya Medical College (Hunan, China). Bovine serum albumin (BSA) was purchased from Shensuo Institute of Biological Products (Shanghai, China). Phosphate-buffered saline (PBS) solutions with various pH values were prepared with 0.01 M Na2HPO4 and 0.01 M KH2PO4. All other reagents were of analytical reagent grade. Doubly distilled water was used throughout the experiments. Fabrication of the Nanogold Particle-Derivatized Surface. The piezoelectric crystals were deposited with plasma-polymerized n-butylamine film following the route reported previously.14-16 (14) Wang, H.; Wang, C. C.; Lei, C. X.; Wu, Z. Y.; Shen, G. L.; Yu, R. Q. Anal. Bioanal. Chem. 2003, 377, 632-638. (15) Wu, Z. Y.; Yong, Y. H.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2000, 412, 29-35.
Main parameters were controlled as follows: the flow rate of pure hydrogen as a carrying gas was 50 mL min-1, the pressure of the reactor chamber was at 266 Pa, and the apparent rf power was 80 W. The PPF-modified crystals were further immersed in the nanogold particle suspensions at 4 °C overnight, then rinsed with water, and dried in air. Preparation of Antibody Fragments. Antibody Fab′-SH fragments were obtained from the controlled reduction of intact leukemic lineage-defined monoclonal antibodies with 2-MEA as described elsewhere.10,11 The reaction conditions for these antibodies were optimized as a 90-min incubation at 37 °C using 2-MEA solution (6 mg mL-1, PBS of pH 6.0) containing 0.15 M NaCl and 5.0 mM EDTA. Then, the low-molecular-weight compounds were removed from the reaction mixture by use of Sephadex G-25 column and by dialysis overnight. The yielded Fab′-SH fragment solutions were either immediately used or stabilized with an equal amount of glycerol to be kept at - 20 °C for future storage within several months. Immobilization of Antibody Fragments. The antibody fragment solutions were premixed in an equal amount according to the defined four leukemic lineages (CD2 and CD7 for T-cell ALL, CD10 and CD19 for B-cell ALL, CD13 and CD33 for granulocyte AML, and CD14 exceptionally with PBS of pH 7.2 for monocyte AML), then separately dropped onto the nanogold particle-derivatized surface of the crystals, and left at 4 °C overnight. After washing with pH 7.2 PBS and water and then drying in air, a 10 mg mL-1 BSA solution was subsequently added onto the crystals to be incubated at 37 °C for 1 h to block the nonspecific binding sites involved. Immunophenotyping Procedure. The prepared four leukemic lineage-associated probes were mounted around the detection vessel containing an assay buffer solution (PBS, pH 7.2, 0.9% sodium chloride). With gentle stirring and thermostatic control at 25 °C, each of the samples to be analyzed was introduced into the detection vessel after stabilization of resonance frequency (shift less than 1 Hz min-1). To avoid the possible error resulting from different additions of samples and deduct the responses induced by nonspecific adsorption, the frequency changes of each probe were recorded as the immunoreaction proceeded from 30 s (after the addition of samples) until equilibrium was reacheds ∼30 min. The control tests with normal (negative) samples and the evaluations for clinical specimens were performed accordingly. The frequency changes in all experiments were referred to the average responses of immunoreaction with corresponding standard deviations (∆F ( SD) of triplicate measurements, unless otherwise indicated. After each immunoassay run, the contaminated QCM crystals were regenerated by rinsing in 8 M urea solution and then washed in the ultrasonic water cleaner, each for 10 min.
by the surface properties of the transducer.13 In this paper, we tried to fabricate an improved interface using nanogold particles and PPF for immobilization of leukocyte-specific antibody fragments onto the piezoelectric probes. Use of nanogold particles was expected to favor a particle-enhanced immobilization of antibodies due to the unique physical and chemical features of nanoparticles.14,17-20 PPF, which possesses some outstanding advantages over the traditional polymer films,14-16,21-22 would be utilized as a desirably robust substrate (matrix) for binding nanogold particles. The fabrication procedures and conditions for the nanogold particle-derivatized interfaces were detailed in our earlier study.14 It is well established that use in combination of multiple leukocyte-specific antibodies, which may respond to the main leukocyte markers existing in the early and later periods or throughout the whole period of acute leukemias, can achieve the identification of acute leukemic blasts from normal cells and the determination of lineages or subsets of acute leukemias.1-3 In this work, the leukocyte-specific monoclonal antibodies were first cleaved into Fab′-SH fragments and then combined (grouped) according to the defined leukemic lineages to be bound onto the nanogold particle-derivatized surface of the probes (CD2 and CD7 for T-cell ALL, CD10 and CD19 for B-cell ALL, CD13 and CD33 for granulocyte AML, and CD14 exclusively for monocyte AML). The four leukemic lineage-associated probes were inserted around the detection vessel that was placed on a magnetic stirrer, and the whole assembly was thermostated by running water of constant temperature through a water pipe. Each of the four aroundmounted probes touched the reaction solution through a small hole, where the attached electric lines of the crystals were extended to a laboratory-made TTL-IC, and further to the highfrequency counters to monitor the induced frequency responses. Figure 1 shows the schematic diagrams of the immunosensor array integrated into the detection vessel. Since the integrated probe array could allow the simultaneous output of multiple phenotypic decision-making parameters with only one analysis of a sample, a time-saving and simplified immunophenotyping for acute leukemia could be thus expected. That is in contrast to the common immunophenotyping methods, i.e., immunofluorescence microscopy,2 where the leukemic lineage-associated information is usually obtained through multiple analysis of a sample using leukocyte-specific antibodies one by one. Moreover, the simultaneous analysis of a sample with the integrated sensors in the same reaction solution might be beneficial to avoid the detection errors resulting from the difference of conditions in one-by-one assays. Immunoreaction Responses of Immobilized Antibodies. A comparative study of the frequency responses of immunoreaction was carried out using antibody fragments immobilized on the probes with and without nanogold particles (bulk gold), where the granulocyte AML samples purified and unpurified (in whole
RESULTS AND DISCUSION Construction of the Integrated Immunosensor Array. As mentioned above, the highly oriented immobilization of antibodies by means of sulfide-terminated antibody fragments presents some superior response performances.10-13 Nevertheless, the activities of the immobilized antibody fragments can be largely influenced
(17) Martin, C. R.; Mitchell, D. T. Anal. Chem. 1998, 70, 322A-327A. (18) Zhao, J. G.; Henkens, R. W.; Stonehuerner, J. L.; O’Daly, J. P.; Crumbliss, A. L. J. Electroanal. Chem. 1992, 327, 109-119. (19) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (20) Grabar, K. C.; Smith, P. C.; Musick, M. D.; Davis, J. A.; Walter, D. G.; Jackson, M. A.; Guthrie, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1148-1153. (21) Muguruma, H.; Karube, I. Trends Anal. Chem. 1999, 18, 62-68. (22) Nakanishi, K.; Muguruma, H.; Karube, I. Anal. Chem. 1996, 68, 16951700.
(16) Yan, Y. H.; Zeng, Y.; Xiang, J. N.; Yin, X.; Jin, J. H.; Zhang, Z. Z. Chin. Sci. Bull. 1998, 43, 1307-1311.
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Figure 2. Comparison of frequency responses of immunoreaction between the probes without and with nanogold particles for immobilizing antibody fragments of granulocyte AML. The probe without nanogold particles was used to detect the (b) purified and (O) unpurified granulocyte AML samples of 5.45 × 104 cells mL-1; the probe with nanogold particles was used to detect the (1) purified and (3) unpurified samples with the same concentration.
blood) were determined in parallel using the two kinds of probes as examples (Figure 2). From Figure 2, one can find that, when detecting purified granulocytes, the probe with nanogold particles shows much greater frequency change (308 ( 23 Hz) than the probe without nanogold particles (221 ( 19 Hz). When detecting the granulocyte sample in whole blood, a higher frequency response of immunoreaction was also observed for the former probe (347 ( 27 Hz) than for the latter probe (282 ( 21 Hz). Moreover, the probe with nanogold particles exhibited more rapid immunoreaction than the probe without nanogold particles in terms of frequency response rate. Some possible explanations may contribute to these observations. First, in contrast to the direct adsorption of proteins (antibodies) on bulk gold surfaces, proteins are bound with well-retained bioactivities on nanogold particlederivatized surfaces of high biocompatibility.17,18,23 Second, the high surface-to-volume area of assembled nanogold particles may greatly enhance the immobilization density of antibodies bound.14,18 Finally, the nanogold particle layer might serve as an intervening “spacer” matrix to extend the immobilized antibody fragments away from the substrate matrix into the mobile phase, resulting in binding sites more accessible to antigens (cells). The “spacer”extended effects have also been confirmed in other immunoassays24 and DNA hybridization tests.25 The above results apparently suggest that the nanogold particle-coated probes with immobilized leukocyte-specific antibody fragments can generate better performances of detecting leukocyte samples both in purified form and in whole blood. Optimization of the Dilution Ratio of Samples. The dilution ratio of samples to be analyzed was optimized through determining (23) Horisberger, M.; Rosset, J. J. Histochem. Cytochem. 1977, 25, 295-305. (24) Ebato, H.; Gentry, C. A.; Herron, J. N.; Mu ¨ ller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. A. Anal. Chem. 1994, 66, 1683-1689. (25) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296.
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various dilutions of leukemic granulocyte and normal (negative) blood samples with the developed granulocyte AML-associated probes. The results are shown in Table 1. As can been seen from Table 1, the frequency changes induced by immunoreacted antigens (granulocytes) increase with the increase of sample dilution in the low dilution range within 1:100 dilution and greatly decrease in the high dilution range beyond the dilution value. Too low or too high sample dilutions might cause serious imbalance in the ratio of antigens to antibodies, resulting in decreased formations of “latticelike” immunocomplex.26 Moreover, the results of examining the normal blood samples as the control tests show that lower sample dilution (higher sample concentration) could cause higher nonspecific adsorption and labile background interference in terms of frequency responses induced. Therefore, the optimal dilution ratio of leukemic samples should have the biggest difference of frequency responses between the leukemic sample and the normal (negative) sample. Such a selection of the optimized dilution ratio is of tremendous importance for obtaining adequate response signal (sensitivity) for clinical decision-making and guaranteeing the reproducibility of system-dependent assays from sample to sample. Obviously, a 1:100 dilution ratio of samples with the peak frequency difference (292 ( 20 Hz) should be recommended in the present experiments. Immunophenotyping Characteristics. The immunophenotyping characteristics of the proposed integrated immunosensor array were studied. Figure 3 displays the typical frequency responses monitored in situ for evaluating the granulocyte AML sample in whole blood. From Figure 3, it can be found that the frequency change for the granulocyte AML-defined probe is much higher than for the other probes, indicating the significant response difference between the lineage-specific recognition and the nonspecific adsorption or crossing recognition. Moreover, the frequency responses among the other three probes are also obviously different, presumably due to the differences of interference degree or crossing-recognition level among lineage-different immunological markers. In addition, the immunophenotyping time was observed to be ∼30 min. Multiple immunophenotyping characteristics were also observed for the evaluation of the T-cell ALL and B-cell ALL samples. When the monocyte AML samples were assessed, however, the frequency responses for the granulocyte AML-associated probe and for the monocyte AML-associated probe were exceptionally approximate. Monocytes, here, as a type of myeloid blasts or myelomonocytes, may simultaneously express the immunological markers of granulocyte AML and monocyte AML (mostly exclusive).1-3,27 Of note, when normal (negative) blood samples were detected using the integrated immmunosensor array as the control tests, all the four kinds of leukemic lineage-associated probes showed substantially lowfrequency responses in contrast to the results obtained when the corresponding leukemic blood samples were assayed. All the immunophenotyping data for the direct assessment of blood samples are summarized in Table 2. As can be seen from Table 2, the frequency response for a lineage-defined sample is 3 times greater than the value for any of the lineage-different or normal blood samples under the same assay conditions. Such a response level for the leukemic samples could be extended as the criterion (26) Atassi, M. Z., van Oss, C. J., Absolom, D. R., Eds. Molecular Immunology; Marcel Dekker: New York, 1984; Chapter 16. (27) Drexler, H. G.; Thiel, E.; Ludwig, W. D. Leukemia 1993, 7, 489-498.
Table 1. Frequency Responses (∆F) to the Normal and Granulocyte AML Blood Samples of Different Dilution Ratios with the Granulocyte AML-Associated Probes dilution ratios of samples
samplesa
∆F1 for granulocyte ∆F2 for normal samplesa ∆F1 - ∆F2
1:10
1:50
1:100
1:150
1:200
1:250
1:300
288 ( 23 68 ( 11 220 ( 12
324 ( 25 61 ( 9 263 ( 16
347 ( 27 56 ( 7 291 ( 20
313 ( 21 43 ( 5 270 ( 16
264 ( 18 36 ( 3 228 ( 15
206 ( 14 29 ( 4 177 ( 10
167 ( 11 22 ( 3 145 ( 8
a ∆F and ∆F are the frequency responses (Hz) presented with mean ( SD (standard deviation) of triplicate measurements for each of the 1 2 blood samples.
Table 2. Frequency Response Results of Immunophenotyping for the Blood Samples of Lineage-Different Acute Leukemias and Normal Human with the Integrated QCM Immunosensor Array
a
ALL samplesa
lineage-associated probes
normal samplea
T-cell
B-cell
T-cell ALL B-cell ALL granulocyte AML monocyte AML
31 ( 5 42 ( 9 56 ( 7 26 ( 4
289 ( 26 47 ( 5 62 ( 9 33 ( 4
53 ( 8 318 ( 26 75 ( 7 46 ( 6
AML samplesa granulocyte monocyte 73 ( 7 85 ( 11 348 ( 27 42 ( 5
35 ( 4 66 ( 9 255 ( 19 283 ( 23
All the frequency responses (Hz) are presented with mean ( SD (standard deviation) of triplicate measurements for each of the blood samples.
Figure 3. Typical immunophenotyping characteristics of frequency responses to the granulocyte AML blood sample (5.45 × 104 cells mL-1) with the QCM immunosensor array composed of (3) the T-cell ALL, (O) the B-cell ALL, (1) the monocyte AML, and (b) the granulocyte AML lineage-associated probes under the optimized conditions.
for judging the immunophenotyping data above which a given phenotype is positively identified or negatively removed. From above discussion, one can conclude that the use of the integrated immunosensor array can readily identify normal cells from leukemic blasts and define the leukemic blasts within certain phenotypic groups (lineages). The utilization of the QCM array system for evaluating the samples in purified sample or in bone marrow could also achieve satisfactory immunophenotyping results (data not shown). Quantitative Analysis. Quantitative analysis of the purified and unpurified granulocyte AML samples was carried out as a model. Figure 4 manifests the plots of frequency change (∆F) versus granulocyte concentration (Ccell). The linear concentration ranges of 5.25 × 103-1.58 × 106 cells mL-1 with detection limit
Figure 4. Calibration profiles of frequency change (∆F) vs granulocyte concentration (Ccell) of the (O) unpurified and (b) purified blood samples of granulocyte AML. Each data point represents the average of the frequency responses of triplicate measurements. Dynamic cell concentration ranges of 0.88 × 104-1.02 × 106 cells mL-1 with detection limit of 5.75 × 103 cells mL-1 and 5.25 × 103-1.58 × 106 cells mL-1 with detection limit of 2.50 × 103 cells mL-1 were observed for the unpurified and purified samples, respectively.
of 2.50 × 103 cells mL-1 and 0.88 × 104-1.02 × 106 cells mL-1 with detection limit of 5.75 × 103 cells mL-1 were obtained for the purified sample and the unpurified one, respectively. Moreover, a purified suspension and an unpurified blood both of 5.45 × 104 cells mL-1 were repeatedly determined each for five times. The relative standard deviation (RSD) among five runs for the former is 8.1% and for the latter, 10.4%. The analytical results for the unpurified blood samples of four kinds of acute leukemias are listed in Table 3. As shown in Table 3, in the case of direct assay of corresponding blood samples, the four kinds of QCM immunosensors exhibit approximately consistent detection ranges of leukocyte concentration exceeding 104-106 cells mL-1, includAnalytical Chemistry, Vol. 76, No. 8, April 15, 2004
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Table 3. Analytical Results for Leukocytic Blood Samples of Lineage-Different Acute Leukemias with the Developed QCM Immunosensors leukocyte types
working ranges (cells mL-1)a
detection limits (cells mL-1)b
detection precision (%)c
T-cell B-cell granulocyte monocyte
1.41 × 104-2.17 × 106 1.13 × 104-1.58 × 106 0.88 × 104-1.02 × 106 1.65 × 104-3.73 × 106
11.30 × 103 7.78 × 103 5.75 × 103 14.05 × 103
12.2 9.7 10.4 11.8
a The working ranges (cells mL-1) are obtained by fitting the calibration curves for determination of acute leukemic blood samples with corresponding QCM immunosensors. b The detection limits (cells mL-1) are estimated according to the σ (standard deviation) rule. c The detection precision values are given out with the RSD among five assay runs.
Figure 5. Equilibrium frequency changes (∆F) vs the number of assay cycles using the granulocyte AML-associated immunosensor, which was renewed with 8 M urea between the successive determinations of the granulocyte blood sample (5.45 × 104 cells mL-1). Average of ∆F of the first 17 assay cycles is 321 ( 35 Hz (mean ( SD)
ing the detection limits and precision values. It is reasonable that the proposed QCM immunophenotyping technique can allow for the directly quantitative determination of acute leukemic samples in whole blood. Reusability of Probes. The regeneration properties of the developed QCM sensing probes were investigated by rinsing the used probes in 8 M urea. Evidence of restoration of the initial resonance frequencies revealed that the dissociation of the antibody-antigen (Fab′-cell) complex was successfully attained by the harsh regeneration procedure. As manifested in Figure 5, up to 17 repetitive assays were achieved without significant loss of detection sensitivity for the as-renewed probe, showing high reusability and stability in the successive assays. Fab′-SH fragments immobilized onto the nanogold particle-derivatized crystal might be considerably stable, and the denaturation or dissociation of the bound proteins in the harsh regeneration process might be postponed owing to the powerful nanometer-sized effects. Moreover, the immobilization surface (matrix) constructed by amine-terminated PPF and nanogold particles might be robust enough to withstand repetitive analysis and regeneration assaults. 2208 Analytical Chemistry, Vol. 76, No. 8, April 15, 2004
Table 4. Immunophenotyping Results for Leukocytic Specimens with Clinically Defined Phenotypes Using the Developed QCM Immunosensor Array QCM array immunoassayb leukocytic phenotypesa T-cell ALL B-cell ALL granulocyte AML monocyte AML normal
specimen T-cell B-cell granulocyte monocyte number ALL ALL AML AML 17 11 26 19 21
16 1 4 1 0
2 11 5 3 2
3 1 23 4 2
1 0 2 17 1
a The leukocytic phenotypes of these specimens were clinically classified using the IEA incorporating with the FCM. b The immunophenotyping results are given out with the number of specimens positively defined for the given lineages of acute leukemia.
The PPF substrate possesses very high mechanical and chemical stability,14-16,21,22 and the assembly of nanogold particles on aminefunctionalized surface may be extremely strong and essentially irreversible.14,19-20 In addition, after ∼17 assay cycles, if the contaminated probes were further treated with the “piranha” solution (H2SO4:H2O2 ) 7:3) to violently peel all the adsorbed organic entities off the surface of the crystals, a complete renewal of the probes could be obtained with reuse lifetime of more than 50 assay runs. Evaluation of Clinical Specimens. To investigate the feasibility of applying the developed QCM immunosensing system for practical immunophenotyping, a number of acute leukemic and normal specimens, which were clinically phenotyped using the IEA and FCM, were evaluated by the proposed QCM immunoassay. The immunophenotyping results are presented in Table 4. From Table 4, one can find that the proposed QCM immunoassay has the probabilities of false positive (6-24%) and false negative (0-12%) phenotypes that are reasonably acceptable for clinical usage. That is, the QCM immunoassay technique could be practically utilized for immunophenotyping of acute leukemia with capabilities comparable to the immunophenotyping methods clinically used. Nevertheless, it is worth noting that the wellrecognized immunophenotyping problem associated with most of the leukocyte blasts (especially the AML ones), frequent expression of multiple makers of different maturation levels and different lineages,1,3,27 might still remain unsolved in the present work. For example, 15-30% leukocytes of AML (granulocyte or momocyte) may express the immunological markers of ALL (T-cell or B-cell).1 Therefore, the complete diagnostic assessments for clinical acute leukemias should be made necessarily based on the comprehensive information of morphology, immunology, cytogenetics, and molecular-genetics, generally known as the MICM working classification for acute leukemia proposed by the World Health Organization (WHO).28 CONCLUSION We have demonstrated here an integrated piezoelectric immunosensor array with viable performances for immunophenotyping of acute leukemia. Some merits of practice lie in the proposed QCM immunosensing system. First, the proposed probe (28) Bennett, J. Int. J. Hematol. 2000, 72, 131-133.
array can analyze the suspected sample once with simultaneous output of multiparameters for phenotypic decision-making, thus allowing a rapid (∼30 min) immunophenotyping of acute leukemia. Second, the orientation-controlled immobilization of antibodies in Fab′-SH fragment fashion, along with the robust sensing interface (matrix) composed of the plasma-polymerized films and nanogold particles, may favor better immunoactivities of antibodies bound and enhanced response performances of sensors. Third, as demonstrated in the evaluation of clinical specimens, the immunophenotyping capabilities of the proposed piezoelectric immunoassay are comparable to those of the clinically used methods in terms of false positive and false negative phenotype probabilities. However, it presents some attractive analytical advantages over the classical analysis formats in that the blood samples may be directly detected without complex purification, labeling, and
separation steps. Finally, the fabrication of the QCM immunosensor array is compatible with micromachining or microfabricating techniques. Therefore, the multiparameter analysis technique may be further automated and miniaturized as a chip-based array for more rapid and high-throughput immunophenotyping of acute leukemia in the future. ACKNOWLEDGMENT This work was supported by the NNSF of China (20075006 and 20375012), the Foundation for Ph.D. Thesis Research (20010532008), and the Science Commission of Hunan Province. Received for review September 19, 2003. Accepted December 23, 2003. AC035102X
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