Selective Detection of HbA1c Using Surface ... - ACS Publications

Jan 22, 2010 - Raman Spectroscopy of Blood and Blood Components. Chad G. Atkins , Kevin Buckley , Michael W. Blades , Robin F.B. Turner. Applied ...
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Anal. Chem. 2010, 82, 1342–1348

Selective Detection of HbA1c Using Surface Enhanced Resonance Raman Spectroscopy Manikantan Syamala Kiran, Tamitake Itoh,* Ken-ichi Yoshida, Nagako Kawashima, Vasudevanpillai Biju, and Mitsuru Ishikawa Nanobioanalysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan In the current work, we report on selective detection of HbA1c, a marker for glycemic control in diabetic patients, using surface enhanced resonance raman spectroscopy (SERRS). We found a characteristic band around 770-830 cm-1 in the SERRS spectrum of HbA1c which was not present in the SERRS spectrum of HbA. To examine the contribution of glucosyl moiety to the characteristic SERRS band of HbA1c, we investigated SERRS spectra for nonenzymatically glycosylated HbA. We found that the SERRS spectral features are essentially identical for both HbA1c and nonenzymatically glycosylated HbA. Furthermore, addition of HbA into colloidal solution of silver nanoparticles (Ag NPs) resulted in the formation of large aggregates of Ag NPs and subsequent sedimentation. On the other hand, aggregation of Ag NPs was considerably low in the case of HbA1c. The differential effect of HbA and HbA1c on colloidal solution of Ag NPs, probably due to their difference in hydrophilicity, enabled us to separate them in a mixture. The separation was characterized by electrophoresis and SERRS analysis. Thus, colloidal solution of Ag NPs and SERRS would be a promising tool for the selective detection of HbA1c. Diabetes, a disorder in the metabolism of glucose due to the lack or inability to use insulin to regulate the glucose level, is one of the major health concerns worldwide.1 High blood glucose has been reported to be the major cause for most of the complications associated with diabetes, such as diabetic nephropathy, diabetic retinopathy, atherosclerosis, and other perivascular diseases.2,3 Diabetes is a disease which is treatable but without any cure; the best possible way to increase the longitivity and decrease the complications associated with it is by proper monitoring of glycemic control.4 The glycemic control refers to the medical term for the level of blood sugar in a diabetic patient, and perfect or good glycemic control means the patient has controlled and maintained the normal glucose levels (70-130 mg/ * Corresponding author. E-mail: [email protected]. (1) Gale, E. A. Diabetes 2001, 50, 217–226. (2) Brownlee, M. Nature 2001, 414, 813–820. (3) Ruderman, N. B.; Williamson, J. R.; Brownlee, M. FASEB J. 1992, 6, 2905– 2914. (4) Hoogwerf, B. J.; Sferra, J.; Donley, B. G. Foot Ankle Clin. 2006, 11, 703– 715.

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dL).5 Glycated hemoglobin level has been used as one of the reliable markers of glycemic control.6-8 Glycated hemoglobin, which is called HbA1c, is formed by nonenzymatic glycosylation of hemoglobin exposed to high plasma levels of glucose.9 The glucose attached to the hemoglobin remains for the life span of red blood cells (RBC, 120 days), indicating that the levels of glycosylated hemoglobin reflect the average blood glucose over 3 months.5 The normal level of glycosylated hemoglobin is less than 6%, and levels beyond 9% indicate poor glycemic control.5 To date, several assays such as high performance liquid chromatography, ion-exchange chromatography, affinity chromatography, isoelectric focusing, boronate affinity chromatography, and immunoassays are available for analyzing glycemic control.8,10-13 However, these techniques present drawbacks with respect to the sensitivity, as the presence of variant hemoglobin and certain clinical factors such as uremia and ethanol ingestion often lead to a faulty result.13 Surface enhanced resonance raman spectroscopy (SERRS) has proven to be a valuable tool for characterizing biomolecules such as heme,14-21 DNA,22 cytochromes,23,24 and pyridine25 because it can increase the Raman cross section by 10-14 orders of (5) Saudek, C. D.; Herman, W. H.; Sacks, D. B.; Bergenstal, R. M.; Edelman, D.; Davidson, M. B. J. Clin. Endocrinol. Metab. 2008, 93, 2447–2453. (6) Koenig, R. J.; Peterson, C. M.; Jones, R. L.; Saudek, C.; Lehrman, M.; Cerami, A. N. Engl. J. Med. 1976, 295, 417–420. (7) Mortensen, H. B. J. Chromatogr., B 1980, 182, 325–333. (8) Bucala, R.; Vlassara, H. Am. J. Kidney Dis. 1995, 26, 875–888. (9) Gallagher, E. J.; Leroith, D.; Bloomgarden, Z. J. Diabetes 2009, 1, 9–17. (10) Trivelli, L. A.; Ranney, H. M.; Lai, H. T. N. Engl. J. Med 1971, 284, 353– 357. (11) Bunn, H. F.; Shapiro, R.; McManus, M.; Garrick, L.; McDonald, M. J.; Gallop, P. M.; Gabbay, K. H. J. Biol. Chem. 1979, 254, 3892–3898. (12) Fisher, R. W.; Dejong, C.; Voight, E.; Berger, W.; Winterhalter, K. H. Clin. Lab. Haematol. 1980, 2, 129–138. (13) Little, R. R.; Goldstein, D. E. Anal. Chem. 1995, 67, 393–397. (14) Podstawka, E.; Proniewicz, L. M. J. Inorg. Biochem. 2004, 98, 1502–1512. (15) Jin, Y.; Nagai, M.; Nagai, Y.; Nagatomo, S.; Kitagawa, T. Biochemistry 2004, 43, 8517–8527. (16) Wang, D.; Spiro, T. G. Biochemistry 1998, 37, 9940–9951. (17) Jayaraman, V.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Science 1995, 269, 1843–1848. (18) Etchegoin, P.; Liem, H.; Maher, R. C.; Cohen, L. F.; Brown, R. J. C.; Milton, M. J. T.; Gallop, J. C. Chem. Phys. Lett. 2003, 367, 223–229. (19) Spiro, T. G.; Strekas, T. C. J. Am. Chem. Soc. 1974, 96, 338–345. (20) Torres Filho, I. P.; Terner, J. J. Appl. Physiol. 2008, 104, 1809–1817. (21) Xu, H.; Bjerneld, E. J.; Kall, M.; Borjesson, L. Phys. Rev. Lett. 1999, 83, 4357–4360. (22) Bell, S. E.; Sirimuthu, N. M. J. Am. Chem. Soc. 2006, 128, 15580–15581. (23) Bailo, E.; Fruk, L.; Niemeyer, C. M.; Deckert, V. Anal. Bioanal. Chem. 2009, 394, 1797–1801. (24) Niki, K.; Kawasaki, Y.; Kimura, Y.; Higuchi, Y.; Yasuoka, N. Langmuir 1987, 3, 982–986. 10.1021/ac902364h  2010 American Chemical Society Published on Web 01/22/2010

magnitude.26,27 SERRS has significantly advanced the detection limit of traditional Raman scattering to be measurable from molecules that would otherwise be spectroscopically silent.28 In SERRS, the target molecule is brought to surfaces or the interface of metal nanoparticles, which enhance both incident and Raman scattering light by coupling with plasma resonance.29-35 The main analytical advantages of SERRS are sensitive and selective detection of molecules due to the distinct vibrational spectrum for each molecule.36 Furthermore, SERRS has many advantages with respect to simple sample preparation, reproducibility, and compatibility with biomolecules.28,37 Thus, SERRS has recently gained applications in biochemical analysis,38 biomedical diagnostics,38 membrane transport processes,39 bacterial cells,40 and intracellular delivery of nanoparticles,40 analysis of colloidal dispersions,22 molecular self-assemblies,22 and oligonucleotides.22 SERRS has been extensively used to understand heme-protein chemistry at ensemble and single molecule levels.14-21 The studies have been extended to single Hb molecule detections.21 Although there are number of reports about the SERRS and HbA,14-21 the SERRS of HbA1c, an important marker for glycemic control has not been investigated in detail. In the current work, we selectively detected HbA1c in the presence of HbA using SERRS. The difference in the aggregation of silver nanoparticles (Ag NPs) by HbA and HbA1c made it possible to separate them in a mixture. Also, we found remarkable spectral differences between SERRS of HbA and SERRS of HbA1c. The presence of a SERRS band at 830 cm-1 for HbA1c was a key to distinguish HbA1c from HbA. We consider that the SERRS band for HbA1c is induced by a glucosyl moiety which was further evidenced by nonenzymatic glycosylation of HbA. Results of the present investigation show that SERRS is a valuable technique for the selective detection of HbA1c. MATERIALS AND METHODS We obtained Hb from Sigma-Aldrich and HbA1c from Lee Biosolutions Inc., and these samples were used as obtained without further purification. Stock solutions of HbA (1.5 × 10-5 M) and (25) Yang, Z.; Li, Y.; Li, Z.; Wu, D.; Kang, J.; Xu, H.; Sun, M. J. Chem. Phys. 2009, 130, 234705. (26) Dieringer, J. A.; Lettan, R. B.; Scheidt, K. A.; Van Duyne, R. P. J. Am. Chem. Soc. 2007, 129, 16249–16256. (27) Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932–9939. (28) Willets, K. A. Anal. Bioanal. Chem. 2009, 394, 85–94. (29) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783–826. (30) Johansson, P.; Xu, H.; Kall, M. Phys. Rev. B 2005, 72, 035427. (31) Xu, H.; Wang, X.; Persson, M. P.; Xu, H. Q.; Kall, M.; Johansson, P. Phys. Rev. Lett. 2004, 93, 243002. (32) Pettinger, B. J. Chem. Phys. 1986, 85, 7442–7451. (33) Itoh, T.; Yoshida, K.; Biju, V.; Kikkawa, Y.; Ishikawa, M.; Ozaki, Y. Phys. Rev. B 2007, 76, 085405. (34) Yoshida, K.; Itoh, T.; Biju, V.; Ishikawa, M.; Ozaki, Y. Phys. Rev. B 2009, 79, 085419. (35) Itoh, T.; Yoshikawa, H.; Yoshida, K.; Biju, V.; Ishikawa, M. J. Chem. Phys. 2009, 130, 214706. (36) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536–1540. (37) Huh, Y. S.; Chung, A. J.; Erickson, D. Microfluid. Nanofluid. 2009, 6, 285– 297. (38) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. Top. Appl. Phys. 2006, 103, 409– 426. (39) Wood, E.; Sutton, C.; Beezer, A. E.; Creighton, J. A.; Davis, A. F.; Mitchell, J. C. Int. J. Pharm. 1997, 154, 115–118. (40) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett. 2006, 6, 2225–2231.

HbA1c (1.5 × 10-5 M) were prepared by dissolving the lyophilized powder in doubly distilled water. These solutions were diluted to required concentrations prior to experiments. A colloidal solution of Ag NPs was prepared by the method described elsewhere.41 The molarity of 1 × 10-11 M and the average particles size of 60 nm was observed for the colloidal solution of Ag NPs, as determined by light scattering analysis. Hb solution was incubated for 3 h with colloidal solution of Ag NPs in the ratio 1:1 at room temperature for adsorption of Ag NPs to Hb molecules. We used 1.5 × 10-6, 1.5 × 10-7, and 1.5 × 10-8 M concentrations of HbA and HbA1c in the present study. An aliquot of incubated hemoglobin solution was spin coated on a glass slide prior to SERRS measurement. The SERRS spectra were measured after incubating HbA and HbAlc molecules with colloidal solution of Ag NPs for 3 h at room temperature (25 °C) for adsorption of HbA and HbAlc molecules to Ag NPs since we found that the SERRS signals were below the detection limit prior to 3 h of incubation, probably due to poor adsorption of HbA1c and HbA on Ag NPs. Experimental Setup. The experimental setup for SERRS is described elsewhere.42 Briefly, a collimated unpolarized white light beam from a 100 W halogen lamp was introduced into an inverted optical microscope (Olympus IX70, Tokyo) through a dark field condenser lens for observing the aggregation of Ag NPs. A cw diode laser (Coherent DPSS 532, Palo Alto, CA; 532 nm, 2 W/cm2) was used as the excitation source. A holographic notch filter was placed behind an objective lens (Olympus LCPlanF1, 60×, N.A. 0.7, Tokyo) to block Rayleigh scattering light. Raman scattering and SERRS imaging was detected using a digital camera (CCD1, Nikkon, COOLPIX5000, Tokyo) and a chargecoupled device (CCD2, DV434-FI, Andor, Tokyo) equipped with a polychromator (Pro-275, Acton, Tokyo) for spectral measurement. Grooves of grating for Raman scattering are usually larger than 1200 L/mm but we used a grating with 150 L/mm grooves in the present investigation to obtain a spectral window with single detection and to avoid temporal spectral fluctuations of SERRS. Absorption and fluorescence spectra of HbA and HbA1c solution (prepared in water) were measured using a U-4100 spectrophotometer (HITACHI) and a F4500 Fluorescence spectrophotometer (HITACHI), respectively. RESULTS AND DISCUSSION Fluorescence and Absorption Spectra of Hb. Figure 1A,B represents the optical absorption and fluorescence spectra of aqueous solutions of HbA and HbA1c. The absorption spectrum of HbA showed characteristic bands at 407, 542, and 588 nm for HbA and at 414, 542, and 574 nm for HbA1c, corresponding to the γ-soret, R, and β bands, respectively. The fluorescence spectrum of both HbA and HbA1c are similar to each other (Figure 1A,B). The R-soret band for HbA and HbA1c is located at around 542 nm. Thus, the excitation at 532 nm resulted in strong fluorescence. Due to strong fluorescence under 532 nm excitation, detection of HbA and HbA1c was difficult (Figure 1C) using conventional Raman spectroscopy. However, detection of HbA and HbA1c by SERRS is possible due to both enhancement (41) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391–3395. (42) Sujith, A.; Itoh, T.; Abe, H.; Anas, A. A.; Yoshida, K.; Biju, V.; Ishikawa, M. Appl. Phys. Lett. 2008, 92, 103901.

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Figure 1. Absorbance and fluorescence spectra of (A) HbA and (B) HbA1c. Inset images represent the absorption spectra of HbA and HbA1c around 500 nm. (C) The resonance Raman spectra of HbA at 532 nm excitation. Strong fluorescence background significantly hinders the Raman measurement.

of Raman scattering light intensity and reduction of fluorescence by energy transfer from hemoglobin to a Ag surface.43 Differentiation of HbA and HbA1c by SERRS Spectra. The SERRS spectrum of HbA showed bands at 1403 and 1652 cm-1, as indicated in Figure 2A. In the case of SERRS of HbA1c, we found a predominant band around 827 cm-1 in addition to the vibrations of porphyrin rings, as indicated in Figure 2B,C. Recently, it has been reported that the SERRS bands of HbA under 514 nm excitation located at 1375, 1586, and 1640 cm-1 and these three prominent bands of Hb molecules represent the vibrations of porphyrin rings in a hemic group packed in the polypeptide chain21,44 However, the band at position 1586 cm-1 was not prominent in our experiments due to spectral overlapping since low energy resolution grating was used in our experiment. (43) Douglas, P.; McCarney, K. M.; Graham, D.; Smith, W. E. Analyst 2007, 132, 865–867. (44) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957–2976.

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Despite the characteristic bands for Hb molecules, we found that the band around 827 cm-1 is a key for distinguishing HbA1c from HbA in the current work. However, it may be noted that the position of this band varied between 770 and 830 cm-1. Note that the major difference between HbA and HbA1c is the presence of a covalently attached glucose moiety in the former. Raman scattering spectra of crystalline and hydrated glucose has been well characterized.45-47 In glucose, Raman scattering bands observed at 820-950 cm-1 are attributed to vibration of υ(C-O), δ(C-C-H), υ(C-C), and δ(C-C-O) bands.45-47 However, glucose does not have optical resonance at the excitation laser wavelength, 532 nm. Thus, the Raman scattering band at 827 cm-1 is not from a free glucose moiety, because the band intensity of glucose should be negligible compared with that of heme; heme provides strong SERRS bands due to its strong optical resonance at this excitation wavelength. In order to further confirm this, we performed SERRS analysis of pure glucose. We found that glucose solution neither caused the aggregation of Ag NPs nor produced the characteristic band under 532 nm excitation, indicating that the contribution of free glucose to the band around 827 cm-1 is negligible. We, thus, consider structural changes in hemoglobin associated with the binding of a glucose moiety generates the band around 827 cm-1 (Figure S1 in the Supporting Information). Indeed, the structural changes in the HbA molecule due to glycation has already been reported.48 Glycation of the HbA molecule reduces R helix content49 and weakens heme-globin interaction.50 To further investigate the origin of this key band between 770 and 830 cm-1, we nonenymatically reacted Hb with glucose and analyzed the SERRS spectrum of the reaction mixture. In order to synthesize nonenymatically glycosylated hemoglobin, we incubated a 1 mg/mL solution of HbA with 8 mg of glucose at room temperature for a week. An aliquot of this mixture (1.5 × 10-6 M Hb) was incubated with colloidal solution of Ag NPs. The supernatant and sediment were separated as described earlier and subjected to SERRS. We observed a characteristic spectral band at 773 cm-1 in case of the supernatant (Figure 3B), compared to sediment (Figure 3A). The pattern of these bands in supernatant and sediment were similar to that observed for HbA1c and HbA, respectively. The appearance of the band at 773 cm-1 in the supernatant indicates the formation of glycosylated hemoglobin due to the nonenzymatic reaction of glucose with hemoglobin. The contribution of free glucose in the supernatant to SERRS was excluded, as the glucose solution did not cause the aggregation of Ag NPs and glucose alone did not produce the characteristic bands under 532 nm excitation. Thus, the appearance of a resonance Raman band at 773 cm-1 suggests that the glucosyl residue attached to the HbA contributed to the characteristic band in SERRS. The formation of glycosylated hemoglobin is reported as a slow and (45) Soderholm, S.; Roos, Y. H.; Meinander, N.; Hotokka, M. J. Raman Spectrosc. 1999, 30, 1009–1018. (46) Barrett, T. W. Spectrochim. Acta, Part A 1981, 37, 233–239. (47) Wells, H. A., Jr.; Atalla, R. H. J. Mol. Struct. 1990, 224, 385–424. (48) Sen, S.; Kar, M.; Roy, A.; Chakraborti, A. S. Biophys. Chem. 2005, 113, 289–298. (49) Cussimanio, B. L.; Booth, A. A.; Todd, P.; Hudson, B. G.; Khalifah, R. G. Biophys. Chem. 2003, 105, 743–755. (50) GhoshMoulick, R.; Bhattacharya, J.; Roy, S.; Basak, S.; Dasgupta, A. K. Biochim. Biophys. Acta 2007, 1774, 233–242.

Figure 2. SERRS of HbA and HbA1c: HbA and HbA1c were incubated with Lee-Meisel Ag colloid and their SERRS spectra measured as described in the Materials and Methods section with excitation at 532 nm and a power of 2 W/cm2 on the sample. (A) The SERRS, dark field image (60×) and SERRS spectrum of HbA (1.5 × 10-6 M). (B) The SERRS, dark field image (60×) and SERRS spectrum of HBA1c (1.5 × 10-6 M). (C) SERRS of HbA and HbA1c. Scale bar represents 10 µm.

nonenzymatic reaction between hemoglobin and glucose.51-53 The rate of this reaction is directly related to the concentration (51) Brownlee, M.; Cerami, A.; Vlassara, H. N. Engl. J. Med. 1988, 318, 1315– 1321. (52) Njoroge, F. G.; Monnier, V. M. In The Maillard Reaction and Aging, Diabetes and Nutrition; Baynes, J. W., Monnier, V. M., Eds.; AR Liss: New York, 1989; pp 85-109.

of glucose. The aldehyde group of glucose reacts with the primary amine group in the hemoglobin and results in the formation of a Schiff base.51-53 The Schiff base is not stable and may either dissociate or undergo further rearrangement, resulting in the (53) Kennedy, L. In International Textbook of Diabetes Mellitus; Alberti, K. G. M. M., DeFronzo, R. A., Keen, H., Zimmet, P., Eds.; John Wiley: Chichester, 1992; pp 985-1007.

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Figure 3. SERRS of HBA incubated with glucose: HBA (1.5 × 10-5 M) (500 µL) was mixed with 8 mg of glucose and kept at room temperature for 1 week. After 1 week, the hemoglobin-glucose mixture was incubated with a colloidal solution of Ag NPs. The supernatant and sediment were collected, and SERRS was measured for both supernatant and sediment. (A) The SERRS spectrum of sediment and (B) the SERRS spectrum of supernatant (HbA). Inset: the representative dark field and SERRS images (60×). Scale bar represents 10 µm.

formation of a stable ketoamine.59-61 Thus, we conclude that appearance of the characteristic band in HbA1c is due to the glucose moiety attached to it. Difference in Aggregation of Ag NPs by HbA and HbA1c. We found that HbA and HbA1c induced aggregation of colloidal solution of Ag NPs. The extent of aggregate formation was detected using SERRS microscopy. The dark field microscopic images of the HbA and HbA1c (Figure 2A,B inset images) shows that HbA induces large Ag NP aggregates compared to HbAlc. The dark field image of Ag NPs aggregate formed by HbA (Figure 2A) appears bright and white but that of HbA1c (in Figure 2B) shows visible colors. The visibility of color is due to plasma resonance indicating that the size of Ag NPs aggregates formed by HbA1c is in the submicrometer scale compared to HbA.56,57 To examine whether the difference in the aggregation of Ag NPs in the presence of HbA and HbA1c is involved in their selective detection, HbA and HbA1c at concentrations of 1.5 × 10-6 M were mixed with colloidal solution of Ag NPs. After (54) Sola, R. J.; Griebenow, K. J. Pharm. Sci. 2009, 98, 1223–1245. (55) Bagger, H. L.; Fuglsang, C. C.; Westh, P. Eur. Biophys. J. 2006, 35, 367– 371. (56) Kuwata, H.; Tamaru, H.; Esumi, K.; Miyano, K. Appl. Phys. Lett. 2003, 83, 4625–4627. (57) Hao, E.; Schatz, G. C. J. Chem. Phys. 2004, 120, 357–366.

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incubation for 3 h, the supernatant of the colloidal solution of Ag NPs was pipetted out from the sedimented aggregates. Aliquots (500 µL) of the supernatant were mixed with an equal volume of colloidal solution of Ag NPs, and the SERRS spectrum was measured. The SERRS spectrum from the supernatant was quite similar to that observed for HbA1c with a characteristic band at 791 cm-1, as indicated in Figure 4B. The SERRS spectrum of the aliquots from the sediment was also measured; the spectrum was similar to that of HbA, and there was the absence of any band in the 770-830 cm-1 region (Figure 4A). To make sure the presence of HbA and HbA1c were within the large and small aggregates of Ag NPs, we subjected the supernatant and sediments to polyacrylamide gel electrophoresis. Here, we used 5% polyacrylamide gel for electrophoresis (Figure 4C). Mild reducing conditions (1% SDS) were used to separate Hb adsorbed to Ag NPs; however, under this reducing condition, we did not observe subunit dissociation of Hb. The gel was stained with coomassie brilliant blue, and the bands were observed at 65 kDa for the sediment and at 68 kDa for the supernatant (Figure 4C). These bands indicate HbA and HbA1c; the molecular weight of HbA and HbA1c is 65 and 68 kDa, respectively.58-60 (58) Adair, G. S.; Bock, A. V.; Field, H., Jr. J. Biol. Chem. 1925, 63, 529–545.

Figure 4. SERRS of HbA and HbA1c mixture: HbA and HbA1c (1.5 × 10-6 M) were mixed and incubated with a colloidal solution of Ag NPs for 3 h. The supernatant and sediment were collected; an aliquot of supernatant was mixed with the same volume of colloidal solution of Ag NPs. SERRS was measured for both supernatant and sediment. (A) The SERRS spectrum of sediment and (B) the SERRS spectrum of supernatant. Inset: the representative dark field and SERRS image (60×). (C) SDS-PAGE (5%) of sediment and supernatant, where M represents protein standard marker, Sed represents the sediment, and Sup represents the supernatant. Scale bar represents 10 µm.

Apparently, the difference in the aggregation of Ag NPs by HbA and HbA1c is related with the glucose moiety attached to it. Glycation of proteins has been reported to affect their hydrophicity and aggregation properties.54,55 The increased solubility upon glycosylation is known to be due to an overall increase in the molecular solvent accessible surface area causing an increase in the number of possible interactions between the protein and the surrounding solvent.54 Further, a decrease in the aggregation has been reported due to glycosylation as a result of an increase in steric repulsion between proteins due to the presence of glycans on the protein surface.54 In addition to this, the net charge of HbA1c is more negative due to the glucose moiety attached on HbA1c compared to HbA. The possibility of the extra negative (59) Adair, G. S. Proc. R. Soc. London, Ser. A 1925, 109, 292–300. (60) Finke, A.; Kobold, U.; Hoelzel, W.; Weykamp, C.; Miedema, K.; Jeppsson, J. O. Clin. Chem. Lab. Med. 1998, 36, 299–308. (61) Goodall, I. Biochem. Rev. 2005, 26, 5–19.

Figure 5. SERRS of HbA and HbA1c: concentration dependence. (A) The dark field and SERRS images of various concentrations of HbA and HbA1c. Panels a-c top to bottom represents 1.5 × 10-6, 1.5 × 10-7, and 1.5 × 10-8 M concentrations of HbA and HbA1c, respectively. Scale bar represents 10 µm. (B) Graphical representation of SERRS spectral intensity from various concentrations of HbA and HbA1c from five different experiments. The SERRS intensity represented is of the heme marker band 1652 cm-1 which is plotted after subtracting the background against respective concentrations of HbA and HbA1c.

charge61 in repulsion of the negatively charged Ag NPs to form aggregates could not be excluded. Thus, we assume that the difference in the aggregation of colloid solution of Ag NPs by HbA and HbA1c may be due to the glycosylation effect.54 The glycosylation of proteins has also been reported to increase the overall stability of protein, preventing chemical instabilities such as proteolytic degradation, oxidation, and chemical cross-linking and physical instabilities like precipitation and pH denaturation.54 Effect of Concentration Variations of HbA and HbA1c on SERRS Spectra. In order to understand the effect of variations in the concentration of Hb molecules on the SERRS spectra, the SERRS spectra for various concentrations of HbA and HbA1c incubated with Ag NPs were measured. We used 1.5 × 10-6, 1.5 × 10-7, and 1.5 × 10-8 M concentrations of HbA and HbA1c in the present investigation. We found that addition of Hb molecules to the colloidal solution of Ag NPs resulted in the aggregation irrespective of the concentration, but the size of the aggregates formed was related with the concentration of hemoglobin. It has been reported that addition of hemoglobin to colloidal solution of Ag NPs results in the formation of aggregates (Figure 5A).21 The size of the aggregates formed with 1.5 × 10-6 M HbA was significantly larger compared to that with 1.5 × 10-8 M (Figure 5A). More importantly, the aggregation of Ag NPs induced by equal Analytical Chemistry, Vol. 82, No. 4, February 15, 2010

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concentrations of HbA and HbA1c was different due to the difference in their hydrophilicity. Also, in the case of HbA1c, a concentration dependent increase in the aggregation of Ag NPs was observed. A concentration dependent variation in the SERRS spectral intensity of HbA and HbA1c is shown in Figure 5B. An electromagnetic field coupled with plasmon of metal nanoaggregates is the origin of SERRS, and the SERRS intensity depends on the electromagnetic field around the metal-molecule aggregate, the number of molecules and hotspots in the aggregates.62,63 In the case of 1.5 × 10-6 M hemoglobin, the relative number of hot spots due to formation of larger aggregates, the electromagnetic field, and number of molecules contributing to SERRS effect is higher compared to 1.5 × 10-8 M hemoglobin, thus resulting in a concentration dependent increase in the SERRS intensity. Representative SERRS spectra at various concentrations of HbA and HbA1c are given as Figure S2 in the Supporting Information. However, it must be noted that there are variations in the SERRS intensity within the same concentration of HbA and HbA1c. The possible reason for observing such variations may be due to the deviation in the molecules adsorbed in Ag NPs; further, the relative number of heme groups contributing to SERRS at different times and at different configurations18 may vary, resulting in such variations in the spectrum.

SERRS as a valuable tool for detecting HbA1c. The present investigation opens up a new strategy for research and development of a SERRS based approach for the detection of HbA1c which may be useful for diabetic diagnosis. The lack of quantitative analysis of HbA1c in the current work could be resolved by the use of standard SERRS active substrates developed recently.64-66 For example, Van Duyne and co-workers have carried out excellent work especially with respect to SERRS measurement of glucose and lactate for the in vivo diabetes diagnosis.67-70 Our findings may open up research for the development of SERRS based sensors for HbA1c as a diagnostic tool in the diagnosis of diabetes.

CONCLUSION In this study, we report selective detection of HbA1c from HbA. The spectrally distinct resonance Raman band at the 770-830 cm-1 position was the origin of this selective detection. The occurrence of this band is attributed to the glucosyl moiety attached to the HbA1c. This was confirmed from the appearance of this band in HbA incubated with glucose. We assume that the difference in the physiochemical properties of HbA1c due to glycosylation resulted in the difference in the aggregation pattern that made it possible to separate HbA1c from HbA in a mixture. Results of the present investigation suggest

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(62) Fang, Y.; Seong, N. H.; Dlott, D. D. Science 2008, 321, 388–392. (63) Imura, K.; Okamoto, H.; Hossain, M. K.; Kitajima, M. Nano Lett. 2006, 6, 2173.

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ACKNOWLEDGMENT The current study was partly supported by “WAKATE B (No. 16760042)” and Priority Area “Strong Photon-Molecule Coupling Fields (No. 470)” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 19, 2009. Accepted January 4, 2010.

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