Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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in addition to their lengthy procedures. 13 Biosensors based on fluorescent (FL) materials have attracted much attention due to their sensitivity, selectivity, and rapidity. Organic molecules and organometallic complexes have been developed for protein assays. 14,15 Upon complexation or conjugation with proteins, the luminophores show emission enhancements and/or spectral shifts, which enable the biopolymers to be detected and quantified. Some of the FL probes, however, are insoluble in aqueous media, while others are unstable under ambient conditions. 16 Some luminophores show spectacular performances in protein assays but are extremely expensive because of their painstaking syntheses. 14,15 These drawbacks greatly limit the scope of their real-life high-tech applications. This calls for the development of synthetically readily accessible and environmentally stable bioprobes with high sensitivity and selectivity. A thorny problem associated with the emissions of conventional luminophores in aqueous medium or physiological buffer is aggregation-caused quenching (ACQ). 17 The luminophores are in close vicinity in the aggregates suspended in the aqueous buffer, which favors the formation of such detrimental species as excimers and exciplexes and causes nonradiative relaxations of the excited states. We have recently discovered that tetraphenylethene (TPE) behaves in a way exactly opposite to the ACQ dyes: it is nonemissive in the solution state but becomes highly luminescent in the aggregate state. 18 We coined “aggregationinduced emission” (AIE) for the phenomenon because the nonemissive TPE is induced to emit by aggregate formation. Decorating TPE with ionic or polar functional groups yields watersoluble derivatives, which can be utilized as FL probes for bioanalyses. 19,20 Our and other research groups have successfully used the TPE-based AIE luminogens for nucleic acid detection, enzymatic activity assay, and metallic ion tracing. 21,22 The successes prompted us to further explore their potentials in bioanaly- (11) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (12) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (13) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Molecular Probe: Leiden, 2002, p. 420. (14) (a) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217. (b) Royer, C. A. Chem. Rev. 2006, 106, 1769. (c) Suzuki, Y.; Yokoyama, K. J. Am. Chem. Soc. 2005, 127, 17799. (d) Matulis, D.; Baumann, C. G.; Bloomfield, V. A.; Lovrien, R. E. Biopolymers 1999, 49, 451. (e) Yarmoluk, S. M.; Kryvorotenko, D. V.; Balanda, A. O.; Losytskyy, M. Y.; Kovalska, V. B. Dyes Pigm. 2001, 51, 41. (f) Hawe, A.; Sutter, M.; Jiskoot, W. Pharm. Res. 2008, 25, 1487. (15) (a) Eryazici, I.; Moorefield, C. N.; Newkome, G. R. Chem. Rev. 2008, 108, 1834. (b) Keefe, M. H.; Benkstein, K. D.; Hupp, J. T. Coord. Chem. Rev. 2000, 205, 201. (16) (a) Matulis, D.; Lovrien, R. Biophys. J. 1998, 74, 422. (b) Davis, D. M.; Birch, D. J. S. J. Fluoresc. 1996, 6, 23. (17) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: London, 1970. (18) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009, 4332. (19) Li, Z.; Dong, Y. Q.; Lam, J. W. Y.; Sun, J.; Qin, A.; Haussler, M.; Dong, Y. P.; Sung, H. H. Y.; Williams, I. D.; Kwok, H. S.; Tang, B. Z. Adv. Funct. Mater. 2009, 19, 905. (20) (a) Yuan, C. X.; Tao, X. T.; Wang, L.; Yang, J. X.; Jiang, M. H. J. Phys. Chem. C 2009, 113, 6809. (b) Zhao, M.; Wang, M.; Liu, H.; Liu, D.; Zhang, G.; Zhang, D.; Zhu, D. Langmuir 2009, 25, 676. (c) Wang, M.; Zhang, D.; Zhang, G.; Zhu, D. Chem. Commun. 2008, 4469. (d) Suzuki, Y.; Yokoyama, K. J. Am. Chem. Soc. 2005, 127, 17799. (21) (a) Tong, H.; Hong, Y.; Dong, Y.; Haussler, M.; Lam, J. W. Y.; Li, Z.; Guo, Z.; Guo, Z.; Tang, B. Z. Chem. Commun. 2006, 3705. (b) Tong, H.; Hong, Y.; Dong, Y.; Haussler, M.; Li, Z.; Lam, J. W. Y.; Dong, Y.; Sung, H. H. Y.; Williams, I. D.; Tang, B. Z. J. Phys. Chem. B 2007, 111, 11817. (c) Hong, Y.; Haussler, M.; Lam, J. W. Y.; Li, Z.; Sin, K. K.; Dong, Y.; Tong, H.; Liu, J.; Qin, A.; Renneberg, R.; Tang, B. Z. Chem.sEur. J. 2008, 14, 6428. 7036 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Chart 1. Chemical Structure of Water-Soluble AIE Luminogen of BSPOTPE ses. In this work, we examined the utility of a TPE salt, sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene(BSPOTPE; Chart 1), as a bioprobe for HSA detection and quantitation. The FL “turn-on” attribute of BSPOTPE by its complexation with albumin facilitated the quantitative assay and visual observation of HSA in the aqueous buffer and gel electrophoresis, respectively. It is well-known that proper biological functions of proteins are associated with their specific strand conformations and folding structures. 23 Understanding of protein folding is of fundamental importance for proteomic and pharmaceutical research. 24 HSA is a polypeptide chain with three R-helical domains (I-III), which are further divided into two subdomains (A and B). 1,25 Its crystal structure shows that the main ligand-binding sites in the albumin are located in the hydrophobic cavities of subdomains IIA and IIIA, which are sometimes referred to as Sudlow sites I and II, respectively. 26 Conformation analyses of the hydrophobic cavities play an important role in drug development, especially pharmacokinetic and pharmacodynamic investigations. 27 Study of conformation transitions of proteins in the presence of denaturants is a topic of great interest because it can offer mechanistic insights into folding and unfolding processes of the biopolymers. 28,29 Though intermediate states have been suspected to be involved in the unfolding and refolding processes of many proteins, they are often not detected due to the lack of appropriate probes. 29 Characterization of the intermediate states becomes even more complex in the multidomain proteins, such as HSA, in which each domain is capable of unfolding and refolding independently. 30 Whether intermediate states are involved in the unfolding pathway of HSA has been an issue of debate. In this study, we made use of the AIE feature of BSPOTPE and investigated the unfolding process of HSA. A stable molten-globule intermediate was observed in its unfolding process induced by guanidine hydrochloride (GndHCl), a well-known denaturant. Förster resonance energy transfer (FRET) study proved the accessibility of HSA by BSPOTPE and suggested probable location of the FL bioprobe in the hydrophobic cavity in the protein folding structure. Combination of the FL technique with circular dichroism (CD), (22) (a) Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. Anal. Chem. 2009, 81, 4444. (b) Wang, M.; Zhang, D.; Zhang, G.; Tang, Y.; Wang, S.; Zhu, D. Anal. Chem. 2008, 80, 6443. (23) Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Bright, F. V. Biophys. J. 1998, 75, 1084. (24) Jusko, W. J.; Gretch, M. Drug Metab. Rev. 1976, 5, 43. (25) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (26) (a) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824. (b) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1976, 12, 1052. (27) Lavinder, J. J.; Hari, S. B.; Sullivan, B. J.; Magliery, T. J. J. Am. Chem. Soc. 2009, 131, 3794. (28) Abou-Zied, O. K.; Al-Shihi, O. I. K. J. Am. Chem. Soc. 2008, 130, 10793. (29) (a) Nolting, B.; Andert, K. Protein Struct. Funct. Genet. 2000, 41, 288. (b) Santra, M. K.; Banerjee, A.; Krishnakumar, S. S.; Rahaman, O.; Panda, D. Eur. J. Biochem. 2004, 271, 1789. (30) Ahmad, B.; Ahmed, M. Z.; Haq, S. K.; Khan, R. H. Biochim. Biophys. Acta Protein Proteomics 2005, 1750, 93.
Figure 1. (A) FL spectra of BSPOTPE in the PBS buffer containing different amounts of HSA. (B) Change in the FL intensity at 475 nm with HSA concentration; I0 ) FL intensity in the absence of HSA. Inset: linear region of the binding isotherm of BSPOTPE to HSA. [BSPOTPE] ) 5 µM; λex ) 350 nm. differential scanning calorimetry (DSC), and molecular modeling helped draw a mechanistic picture on the protein unfolding process. MATERIALS AND METHODS General Information. BSPOTPE was prepared according to our previously published procedures. 21b HSA, GndHCl, and other biomolecules were all purchased from Sigma and used as received. Phosphate buffered saline (PBS) with pH of 7.0 was purchased from Merck. Water was purified by a Millipore filtration system. All the experiments were performed at room temperature unless otherwise specified. UV spectra were measured on a Milton Roy Spectronic 3000 Array spectrophotometer, and FL spectra were recorded on a Perkin-Elmer LS 55 spectrofluorometer with a Xenon discharge lamp excitation. CD spectra were recorded on a Jasco J-810 spectropolarimeter in a 1 mm quartz cuvette using a step resolution of 0.2 nm, a scan speed of 100 nm/min, a sensitivity of 0.1°, and a response time of 0.5 s. Each spectrum is the average of three scans. Details about the artificial urine preparation, cytotoxicity assay, FRET study, DSC and pH-dependent FL measurements, and computation modeling are given in the Supporting Information. Sample Preparation. Stock solutions of BSPOTPE and HSA with a concentration of 1.0 mM were prepared by dissolving appropriate amounts of the luminogen and protein in the PBS buffer. The final concentration of HSA in PBS was double checked by measuring its absorbance at 279 nm. In the HSA unfolding study, HSA (0.4 µM) in PBS was incubated in the presence of different amounts of GndHCl (0.2-7.0 M) at 25 °C for 30 min. Afterward, BSPOTPE was added to the mixtures and incubated for another 30 min before spectral measurements. FL spectra were recorded in the wavelength range of 370-670 nm using 350 nm as the excitation wavelength. For the intrinsic tryptophan fluorescence study, FL spectra were collected from 310 to 570 nm using 290 nm as the excitation source. In the refolding experiment, 0.2 mM HSA was incubated in 8 M GndHCl for 24 h at 25 °C to ensure that all the proteins were fully unfolded. The denatured sample was then diluted with PBS until the final concentrations of HSA and GndHCl were equal to 0.4 µM and less than 0.1 M, respectively. Fraction of refolded proteins (F r) was calculated from the following equation: F r ) 1 - I N - I R I N - I D where IN and ID are the FL intensities of native and denatured HSA-BSPOTPE complexes, respectively, and IR is the FL intensity of BSPOTPE recovered from the refolded HSA. Electrophoresis Assay. A poly(acrylamide) gel electrophoresis (PAGE) experiment was performed on a Hoefer miniVE system under nondenaturing conditions using 5% stacking and 12% resolving native poly(acrylamide) gel at 100 V for 3hat4°C. After electrophoresis, the gel was soaked in an aqueous solution of BSPOTPE (1 mg in 100 mL ddH2O) at room temperature for 5 min. Alternatively, the gel was stained with a Coomassie Brilliant Blue (CBB) solution (0.1% w/v Coomassie Blue R250 in an aqueous mixture containing 10% methanol and 7% acetic acid) for6htoovernight on a rotary shaker with gentle mixing, followed by destaining in an aqueous solution containing 10% methanol and 7% acetic acid for 1 to 2 h until the background of the gel became transparent. An AlphadigiDoc system with a DE-500 MultiImage II light cabinet and an ML-26 UV transilluminator (Alpha Innotech) was used for data collection and analysis. RESULTS AND DISCUSSION Protein Quantitation in Solution. BSPOTPE is soluble in water but insoluble in common organic solvents, such as acetonitrile, THF, and chloroform. Its FL quantum yield (ΦF) is increased from 0.37% in water to 17.5% in acetonitrile, where the luminogen molecules aggregate. The BSPOTPE solution in PBS is feebly luminescent at 390 nm in the absence of HSA (Figure 1A). When a small amount of HSA is added, the BSPOTPE solution becomes luminescent. Its FL intensity at 475 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 (1) 7037
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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of which was constant for all ampli
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
Page 245 and 246: Since the time for liquid expulsion
Page 247 and 248: Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
Page 253 and 254: Figure 4. (A) Typical raw electroph
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Page 257 and 258: Another approach to handle this lim
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Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: CONCLUSIONS In this paper we have i
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Page 267 and 268: Figure 2. Qdot-based FLFTS response
Page 269 and 270: Figure 6. Fluorescence imaging of Q
Page 271 and 272: Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274: Table 1. Effect of 1 D-LC Condition
Page 275 and 276: Figure 3. Peak-production rate vers
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Page 279 and 280: luciferase (T7 control vector) was
Page 281 and 282: Figure 3. Inhibitory effects of luc
Page 283 and 284: Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286: Electrochemistry. Electrochemical m
Page 287 and 288: Figure 2. SEM images of Au substrat
Page 289 and 290: Table 3. Film Thickness Measurement
Page 291: Anal. Chem. 2010, 82, 7035-7043 Qua
Page 295 and 296: Figure 4. (A) Variation in the FL i
Page 297 and 298: Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300: Scheme 1. Proposed Mechanism for Fl
Page 301 and 302: one or more drawbacks including poo
Page 303 and 304: Figure 2. Free fluorophores do not
Page 305 and 306: Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308: Figure 2. Comparative analysis of d
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