[雙語(yǔ)翻譯]---醫(yī)學(xué)外文翻譯--超靈敏免疫和dna電化學(xué)生物分析（原文）

1、Ultrasensitive Electrical Biosensing of Proteins and DNA: Carbon-Nanotube Derived Amplification of the Recognition and Transduction EventsJoseph Wang,* Guodong Liu, and M. Rasul Jan?Department of Chemistry and Biochemist

2、ry, New Mexico State UniVersity, Las Cruces, New Mexico 88003Received December 15, 2003; E-mail: joewang@nmsu.eduThe detection of DNA and proteins is of central importance to the diagnosis and treatment of genetic diseas

3、es, to the detection of infectious agents, drug discovery, or warning against bio-warfare agents.1-4 Such biodetection commonly relies on hybridization or antigen-antibody (Ag-Ab) interactions, and requires proper at- te

4、ntion to the achievement of ultrasensitive measurements. Elec- trochemical transducers are very attractive for such bioassays, owing to their high sensitivity, inherent simplicity and miniaturization, and low cost and po

5、wer requirements. The use of enzyme labels to generate electrical signals has been extremely useful for ultrasen- sitive electrochemical bioaffinity assays of proteins and DNA. Heller’s group5,6 demonstrated that a highl

6、y sensitive amperometric monitoring of DNA hybridization (down to 5 zmol) could be achieved in connection with a horseradish-peroxidase (HRP)-labeled target and an electron-conducting redox polymer. HRP label has been co

7、mbined by Willner’s group7,8 with a biocatalytic precipitative accumulation of the enzyme-generating product to achieve multiple amplifications and very low (25 amol) detection limits. Efforts to amplify enzyme-linked el

8、ectrical protein assays included dual- enzyme substrate recycling9 or ion-exchange accumulation of the product.10 Yet, amplified transduction of biological recognition events remains a major challenge to electrical bioas

9、says. New schemes based on coupling the biocatalytic amplification of enzyme tags with additional amplification units and processes are highly desired for meeting the high sensitivity demands of electrochemical detection

10、 of proteins and nucleic acids. Here we demonstrate the use of carbon nanotubes (CNTs) for dramatically amplifying enzyme-based bioaffinity electrical sensing of proteins and DNA. The unique electronic, chemical, and mec

11、hanical properties of CNTs make them extremely attractive for electrochemical sensors.11,12 Most CNT-sensing work has focused on the ability of surface-confined CNTs to promote electron-transfer reactions involved in bio

12、catalytic devices.13,14 In our new bioaffinity assays (Figure 1), CNTs play a dual amplification role in both the recognition and transduction events, namely as carriers for numerous enzyme tags and for accumulating the

13、product of the enzymatic reaction. These novel support and preconcentration functions of CNTs reflect their large specific surface area15 and are illustrated using the alkaline phosphatase (ALP) enzyme tracer. Such coupl

14、ing of several CNT-derived amplification processes leads to the lowest detection limit reported thus far for electrical DNA detection. The new CNT-based amplified bioelectronic protocol (Figure 1) involves the sandwich h

15、ybridization (a) or antigen-antibody (b) binding along with magnetic separation of the analyte-linked magnetic-bead/CNT assembly (A), followed by enzymatic ampli- fication (B), and chronopotentiometric stripping detectio

16、n of the product at the CNT-modified electrode (C). Our TEM observations (e.g., Figure 2) indicate that the hybridization event leads to cross linking of the ALP-loaded CNTs and the magnetic beads (with theDNA duplex act

17、ing as “glue”). To our knowledge, this is the first example of using DNA for linking particles to CNTs. No such aggregation was observed in the presence of noncomplementary oligonucleotides (B). Apparently, without the r

18、ecognition event, the ALP-tagged CNTs are removed by the magnetic separation, leaving the magnetic beads behind. ALP was immobilized on CNTs using a 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide linker (see Figure 1 in

19、Supporting Information). A coverage of around 9600 enzyme molecules per a CNT (i.e., binding event) was estimated from a separate electrochemical experiment comparing the R-naph- thol response of known amounts of ALP-loa

20、ded CNTs and ALP (assuming similar activities for the free and bound ALP). The dramatic signal enhancement associated with the CNT-based dual amplification route is demonstrated in Figure 3 for DNA- hybridization (A) and

21、 Ag-Ab (B) bioassays. The conventional protocols, based on the single-enzyme tag and a glassy-carbon transducer, are not responding to either 10 pg mL-1 DNA target (A,a) or 80 pg mL-1 IgG (B,a). The first amplification s

22、tep based on the ALP-loaded CNTs (b) offers convenient detection of these low analyte concentrations. The single-ALP protocols displayed a? Permanent address: Department of Chemistry, University of Peshawar, Pakistan.Fig

23、ure 1. Schematic representation of the analytical protocol: (A) Capture of the ALP-loaded CNT tags to the streptavidin-modified magnetic beads by a sandwich DNA hybridization (a) or Ab-Ag-Ab interaction (b). (B) Enzymati

24、c reaction. (C) Electrochemical detection of the product of the enzymatic reaction at the CNT-modified glassy carbon electrode; MB, Magnetic beads; P, DNA probe 1; T, DNA target; P2, DNA probe 2; Ab1, first antibody; Ag,

25、 antigen; Ab2, secondary antibody; S and P, substrate and product, respectively, of the enzymatic reaction; GC, glassy carbon electrode; CNT, carbon nanotube layer.Figure 2. TEM images of the magnetic beads-DNA-CNT assem

26、bly produced following a 20-min hybridization with the 10 (A) and 0 (B) pg mL-1 target sample. The micrographs were taken with a Hitachi H7000 instrument operated at 75 kV.Published on Web 02/18/20043010 9 J. AM. CHEM. S

27、OC. 2004, 126, 3010-3011 10.1021/ja031723w CCC: $27.50 © 2004 American Chemical Societylower signal for a significantly (1000-fold) higher target concentra- tion (not shown). The nearly 104 improvement in the sensit

28、ivity is in good agreement with the estimated ALP loading per CNT. Only ～50-fold sensitivity enhancement was observed by using a strepta- vidin-coated polystyrene carrier bead instead of the CNT support. Further enhancem

29、ents of the DNA and protein signals (by ～30- fold) are observed in the second amplification path, employing the CNT-modified transducer (c). The latter reflect the strong adsorptive accumulation of the liberated R-naphth

30、ol on the CNT layer. The preconcentration feature of the CNT layer was indicated from the use of different accumulation times that led to a sharp increase in the R-naphthol signal (compared to the time-independent signal

31、 observed at the bare electrode; see Figure 2 in Supporting Information). Figure 4A displays typical chronopotentiograms for extremely low target DNA concentrations (0.01-100 pg mL-1; a-e). Well-defined R-naphthol signal

32、s are observed for these low DNA concentrations in connection with 20-min hybridization. The resulting plot of response vs log[Target] (shown as inset) is linear and suitable for quantitative work. The favorable response

33、 of the 5 fg mL-1 DNA target (B) indicates a remarkably low detection limit of around 1 fg mL-1 (54 aM), i.e., 820 copies or 1.3 zmol in the 25 µL sample. Such a low detection limit compares favorably with the lowes

34、t values of 5 zmol (3000 copies) and 25 amol reported for electrical DNA detection.6,8 Similarly, IgG was determined with a detection limit of 500 fg mL-1 (160 zmol in 25 µL samples) and exhibits a well-defined loga

35、rithmic concentration dependence. The smaller signal observed in a control experiment for a huge (～106) excess of a noncomplementary oligonucleotide (Figure 4, C vs B) reflects the high selectivity associated with the ef

36、fective magnetic separation. The amplified electrical signal is coupled to a good reproducibility. Two series of six repetitive measurements of 1 pg mL-1 DNA target or 0.8 ng mL-1 IgG yielded reproducible signals with re

37、lative standard deviations of 5.6 and 8.9%, respectively. In conclusion, we have demonstrated a CNT-based dual ampli- fication route for ultrasensitive electrical bioassays of proteins and DNA. The use of CNT amplifiers

38、(loaded with numerous ALP tags) has been combined with the preconcentration feature of CNT transducers to yield a dramatic enhancement of the sensitivity. Such coupling of several CNT-derived amplification processes resu

39、lts in highly sensitive detection of proteins and DNA and hence indicates great promise for PCR-free DNA assays. Further improve- ments in the sensitivity are expected either through reducing the electrode size and sampl

40、e volume6 or by substrate recycling.9 The new CNT-derived amplification bioassays are expected to open new opportunities for medical diagnostics and protein analysis. The finding that DNA hybridization can be used for li

41、nking CNTs to particles holds promise for assembling controllable nanoscale systems.Acknowledgment. Financial support from the National Science Foundation (Grant CHE 0209707) and National Institutes of Health (Award R01A

42、 1056047-01) is gratefully acknowledged.Supporting Information Available: Related experimental condi- tions (instrumentation, reagents, sequences, and procedures) along with additional data (PDF). This material is availa

43、ble free of charge via the Internet at http://pubs.acs.org.References(1) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A. (2) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192. (3) Wang, J. Che

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46、A.; Halsall, H. B.; Heineman, W. R.; Scheller, F. W. Anal. Chem. 1996, 68, 2453. (10) Limoges, B.; Degrand, C. Anal. Chem. 1996, 68, 4141. (11) Baughman, R. H.; Zakhidov, A.; de Heer, W. A. Science 2002, 297, 787. (12) Z

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48、Laurent, C.; Flahaut, E.; Basca, R.; Rousset, A. Carbon 2001, 39, 507.JA031723WFigure 3. Chronopotentiometric signals for 10 pg mL-1 target oligonucle- otide (A) and 80 pg mL-1 IgG (B) using the glassy carbon (GC) transd

49、ucer and (a) a single ALP tag and (b) CNT-loaded with multiple ALP tags; (c) same as (b) but using the CNT-modified GC electrode. Amount of magnetic beads, 50 µg; sandwich assay with 20 and 30 min for each hybridiza

50、tion event and Ag/Ab association, respectively; sample volume, 50 µL. Detection, addition of 50 µL R-naphthyl phosphate (50 mM) solution with a 20-min enzymatic reaction. Measurements of the R-naphthol product

51、were per- formed at the bare or modified GC electrodes, using a 2-min accumulation at +0.2 V in a stirred phosphate buffer solution (0.05 M, pH 7.4; 1 mL), followed by a 10-s rest period (without stirring) and applicatio

52、n of an anodic current of +5.0 µA. See Supporting Information for the concentrations of the oligonucleotide probes and antibody, and sequence of oligonucleotide probes, levels and preparation of the ALP-DNA-CNT and

53、ALP- streptavidin-CNT conjugates.Figure 4. Chronopotentiometric signals for increasing levels of the DNA target: (a) 0.01, (b) 0.1, (c) 1, (d) 50, (e) 100 pg mL-1. Also shown (inset) is the resulting calibration plot (A)

54、, and the response for 5 fg mL-1 target DNA (B) and 10 ng mL-1 noncomplementary (NC) oligonucleotide (C). Sample volume, 25 µL (B) and 50 µL (C). Other conditions, as in Figure 3 (A,c) based on protocol of Figu