Presentation is loading. Please wait.

Presentation is loading. Please wait.

Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI National Institute of Advanced Industrial Science and Technology.

Similar presentations


Presentation on theme: "Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI National Institute of Advanced Industrial Science and Technology."— Presentation transcript:

1 Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI National Institute of Advanced Industrial Science and Technology (AIST), Japan November 29 th -30 th, 2010 NANOJASP2010 Barcelona, Spain Contents 1. Introduction 2. Approach based on probe conformation 3. Approach based on signal-generating/suppressing moieties 4. Summary 1

2 Mechanism of Biological Effect body cell environment nucleolus transciption biological effect chemicals gene expression proteinsenzymes Mechanism of Biological Effect of Chemical Substances Gene expression allows to evaluate biological effect at a genetic level and to collect genetic information rapidly without animal tests. DNA microarray is widely used in a laboratory level as a comprehensive diagnosis tool for gene expression in medical and environmental fields. mRNAs DNA microarray extraction chemical exposure cell 2

3 A conventional technique of oligonucleotide detection --- Spectroscopic technique based on fluorescence labeling Background DNA microarray - Time-consuming and expensive process - Affection to quantitativity - Needing a wash for removing unbound targets Present problems fluorescent label Hybridization probe h Probe array target DNA microarray scanner (company A) Size: 60 x 90 x 60 [cm] Example We have studied on these problems by electrochemical approaches, expected to contribute simplifying and size-reducing the detection system. We have studied on these problems by electrochemical approaches, expected to contribute simplifying and size-reducing the detection system. 3 solid surface

4 Approach based on electrostaticts Hybridization changes the surface charge, inhibiting redox reaction of the marker, enables label-free target detection. Approach based on probe conformation Hybridization makes the probe structure more rigid, inhibiting redox reaction of Fc, enables label- and reagent-free target detection (self-reporting DNA detection). Approach based on signal-generating/suppressing moieties Hybridization makes the probe structure more rigid, restoring redox reaction of Fc, enables self- reporting and “signal-on” target detection (“OFF”→“ON”). Approaches ONOFF ONOFF ONOFF Simple and rapid electrochemical gene detection techniques signal-generating moiety: Fc signal-suppressing moiety: β-CD electroactive moiety: Fc electroactive marker: [Fe(CN) 6 ] 4- 4

5 Scheme of electrochemical experiments Electrochemical measurement - cyclic voltammetry (CV) - square wave voltammetry (SWV) target DNAs in 0.1 M NaClO mM phosphate buffer (Na +, pH 7.0) in 0.1 M NaClO mM phosphate buffer (Na +, pH 7.0) Ag/AgCl reference electrode Pt auxiliary electrode Modification Incubation Measurement after incubation for 20 min, at 65 o C, cooling down to 25 o C 5’GCA ACC TTC CCT ATT ACT CCA C 3’ 3’CGT TGG AAG GGA TAA TGA GGT G 5’ 3’ATG ACA CCA ATA ACG ACA GA 5’ Fc-PNA: DNA_1: DNA_2: probe Fc-PNA complementary to Fc-PNA mismatched to Fc-PNA Sequences for the probe and targets 5 Approach based on probe conformation anchorpeptide nucleic acid (PNA)signal 5’3’ probe solution electrode - probe: 0.1 mM Fc-PNA for 2 h - thiol: 1 mM 11-HUT for 12 h Fc-PNA 11-HUT

6 Evaluation of probe surface density based on electron transfer reaction Fc-PNA gold electrode CV for the electrode in 0.5 M KOH CV Scan rate: 0.1 V s -1 S-Au: 32.9 pmol cm -2  surface density of S-Au bonds RS–Au + e – RS – + Au 0 CV for the electrode in a buffer solution CV Scan rate: 0.01 V s -1 Fc: 33.1 pmol cm -2  surface density of Fc Fc 0 Fc + + e - Every probe is immobilized on the surface, keeping its Fc moiety. 6 Electrochemical characterization

7 diffusion-like motion surface-confined motion Change in probe flexibility — before hybridization Scan rate: 0.2 V s -1 – 51.2 V s -1 CVs EpEp Current · (Scan Rate) -1 / µA s V -1 Potential / V vs Ag/AgCl Dependence of CVs on scan rate (DNA_1 = 0 M) More irreversible CVs were measured at higher scan rates, indicating the Fc moiety needs more time for diffusion to cause electron transfer. peak potential separation,  E p, vs scan rate, v Scheme 1: Thermal vibration * H. Aoki and H. Tao, Analyst 2007, 132, Access to the surface proceeds to Fc redox reaction bulk surface 7 Change in probe flexibility

8 Plot of log(anodic peak current, i pa ) vs log(scan rate, v) slope ~1 slope ~1/2 diffusion-like motion surface-confined motion From slopes in the plot of log i pa vs log v,  surface-confined motion  diffusion-like motion slope ~1/2,2. slope ~1,1. 1 The change in the Fc character revealed that the Fc moiety is located at the loose end of the probes, subject to thermal vibration. vcvc CVs i pa Current · (Scan Rate) -1 / µA s V -1 Potential / V vs Ag/AgCl Change in probe flexibility — before hybridization Dependence of CVs on scan rate (DNA_1 = 0 M) * H. Aoki and H. Tao, Analyst 2007, 132, Change in probe flexibility

9 change in flexibility M 0 M M change in flexibility Upon hybridization, the value of scan rate at which the motion changes (v c ) was shifted to be lower. This suggests the decrease in probe flexibility.  Detection of target DNAs using this change in Fc character Plot of log(anodic peak current, i pa ) vs log(scan rate, v) Change in probe flexibility — after hybridization Peak potential separation vs scan rate (DNA_1 = 0 M) e–e– before hybridization after hybridization Scheme 2: Change in probe flexibility 9 Approach based on probe conformation

10 DNA concentration dependence Dependence of CVs on DNA concentration Dependence of SWV on DNA concentration M of DNA_1 0 M M DNA_1 0 M SWVs Step potential: 2 mV Amplitude: 25 mV Frequency: 50 Hz i o – i baseline i – i baseline CVs (i – i baseline ) / (i o – i baseline ) DNA_2 (mismatch): 10 –4 M DNA_1 (complementary) Detection limit: 1.4 x 10 –11 M Sensor response dependence on target concentration (SWV) Detection limit: 1.4× M (S/N = 3.0)  Sequence-specific DNA detection was achieved  based on the change in the probe flexibility  without labeling targets nor adding external  markers (“self-repoting”). Scan rate: 1 V s -1 * H. Aoki and H. Tao, Analyst 2007, 132, Hybridization with target DNAs

11 Repeated use of the prepared DNA sensors Regeneration of the sensors rehybridization (10 -4 M of DNA_1) denaturation SWVs Step potential: 2 mV Amplitude: 25 mV Frequency: 50 Hz Relative change in peak currents in regeneration process - 1…1 st measurement (right after prep.) - 2, 4…1 st and 2 nd hybridization DNA_1, 10 –4 M - 3, 5…1 st and 2 nd denaturation ( in 2 M urea, 65 o C ) - 6…mismatched DNA_2, 10 –4 M 1 st measurement DNA_1, 10 –4 M DNA_1, 10 –4 M denaturation DNA_2, 10 –4 M The electrodes modified with Fc-DNA monolayer can be used repeatedly. * H. Aoki and H. Tao, Analyst 2007, 132, Repeated use of the sensors

12 Molecular beacon (PNAS, 2003, 100, 9134 (Plaxco et al.)) Change in flexibility (JACS, 2003, 125, 1112 (Anne et al.)) Electron wire (PNAS, 2005, 102, (Inouye et al.)) Aptamer (Angew. Chem., 2005, 44, 5456 (Plaxco et al.)) Aptamer (JACS, 2006, 128, 117 (O’Sullivan et al.)) “Self-reporting” probes from other research groups Detection limit: 10 pM Detection limit: 5 µM Detection limit: 100 µM Detection limit: 6.4 nM Detection limit: 0.5 nM Change in flexibility (Analyst, 2007, 132, 784 (Aoki et al.)) “ON” “OFF” DNA Fc-PNA Detection limit: 14 pM Improvement of sensitivity  due to high Tm and flexibility in PNA 12 Other contemporary probes Almost of all reported probes were based on a “signal-off” architecture.

13 probe Hybridization electrode “OFF” “ON” e–e– Approach based on a “signal-on” architecture Development of label- and reagent-free (self-reporting), and “signal-on” probes. Development of label- and reagent-free (self-reporting), and “signal-on” probes. Patent application: -JP patent application , , , Hybridization “ON” “OFF” Fc e–e– marker : [Fe(CN) 6 ] 4– Hybridization “ON”“OFF” e–e– Label-free Reagent-free Self-reporting Self-report&“signal-on” Suppressing redox activity “OFF” Inclusion complex β-cyclodextrin ferrocene Restoring redox activity “ON” dissociation e–e– Hybridization DNA “Signal-on” architectures have advantage of higher sensitivity over “signal-off” ones. 13 Improvement of sensitivity for DNA detection The use of probes emitting signals upon hybridization, i.e., “signal-on” probes, is important.

14 Approach based on a “signal-on” architecture Scheme of electrochemical experiments pmol, 5 µL CD-DNA-Fc in 7.5 mM NaCl + 75 mM phosphate buffer (Na +, pH 7.0) Measurement Electrochemical measurement --- cyclic voltammetry (CV) scan rate: 0.01 V s -1 Interdigitated array electrode (carbon) Width: 10 µm Gap: 5 µm Length: 2 mm Number: 65 5’GCA ACC TTC CCT ATT ACT CCA C 3’ 3’CGT TGG AAG GGA TAA TGA GGT G 5’ CD-DNA-Fc: DNA: CD-DNA-Fc (22 mer) complementary to CD-DNA-Fc Sequences of the probe and target 14 e–e– e–e– Fc hybridization electrode Detection system using probes without anchors enables detection in bulk solutions. CD-DNA-FC

15 Potential, E / V Current, I / nA Potential, E / V Current, I / nA Results Dependence of CVs on target concentration 12.5 pmol 0 mol CVs e-e- e-e- Ferrocene moiety ・ Redox potential: negative shift of Δ62 mV ・ Current change: 1.3 nA  6.9 nA (5-fold)  The redox activity of Fc was restored Potential / V vs AgAgCl Current / µA Step E: 2 mV Amplitude: 25 mV Frequency: 50 Hz SWVs e-e- OH e-e- [ref.] SWV of HMFc ( 0.3 mM ) + β-CD ( 15 mM ) 12.5 pmol 0 mol Article: Supramol. Chem., 22, 455 (2010) 15 The probe works based on a self- reporting “signal-on” architecture.

16 Summary 16 e–e– e–e– Fc hybridization electrode “ON” “OFF” 1. Based on conformational flexibility change in probe structure, label-free and reagent-free (“self-reporting”) DNA detection was achieved. 2. Based on signal-generating and suppressing moieties in a probe, “self-reporting” and “signal-on” DNA detection was achieved. These approaches are expected to contribute to more simple and rapid DNA detection. These approaches are expected to contribute to more simple and rapid DNA detection.

17 Tsukuba, 45 min from Tokyo Mt. Tsukuba Tsukuba Express (TX), established in 2005 National Institute of Advanced Industrial Science and Technology (AIST) Japan Aerospace Exploration Agency (JAXA) National Institute for Materials Science (NIMS) University of Tsukuba At last … What is AIST? 17

18 Acknowledgements Collaborators - Prof. Emeritus Yoshio UMEZAWA (Univ. Tokyo) - Prof. Masao SUGAWARA (Nihon Univ.) - Prof. Koji TOHDA (Univ.Toyama) - Prof. Philippe BUHLMANN (Univ. Minnesota) - Prof. Sandra RONDININI (Univ. Milan) - Prof. Marcin MAJDA (UC Berkeley) - Dr. Hiroaki TAO (AIST) - Dr. Masaki TORIMURA (AIST) - Dr. Hiroaki SATO (AIST) - Dr. Hanna RADECKA (Polish Academy of Science) - Akiko KITAJIMA, Tsutomu FIJIKAKE Financial Supports - MEXT: Grant-in-Aid for Young Scientists (B) (Nos , ) Grant-in-Aid for Scientific Research Innovative Areas (No ) - JST: Research Grant for Promoting Technological Seeds (No ) Thank you for your attention! 18

19

20 Chemicals Used in This Study covalent bond S Au gold disk electrodes* 1. probe: 0.1 mM Fc-PNA for 2 h 2. thiol: 1 mM 11-HUT for 12 h * gold disk electrodes geometric surface area: 2.01 mm 2 (  1.6 mm) real surface area: 4.22 mm 2 (from CV in 1 M H 2 SO 4 ) surface roughness factor: 2.10 solutions - Molecules are immobilized on the surface via S-Au bonds. - Thiols prevent the probe from non-specific adhesion. 5’3’ anchorpeptide nucleic acid (PNA) signal 20 Approach based on probe conformation

21 Conclusion Hybridization at probe monolayers changes in the probe flexibility and inhibits redox reaction of Fc. Fc-PNA allows label-free and marker-free sequence-specific DNA detection as a “self-reporting” probe. “ON” “OFF” SWV M 0 M Step potential: 2 mV Amplitude: 25 mV Frequency: 50 Hz Detection limit: 1.4×10 –11 M Target DNA Fc-PNA Approach based on probe conformational flexibility * H. Aoki and H. Tao, Analyst 2007, 132, Conclusion

22 Potential / V vs Ag/AgCl Current / µA 0 M DNA_1: 10 –4 M i bi b i ai a CVs Scan rate: 0.1 V s -1 Signal amplification by using electron donors Fc Fc + + e – Fc + + [Fe(CN) 6 ] 4– Fc + [Fe(CN) 6 ] 3– 1 – i a /i b = Potential / V vs Ag/AgCl Current / µA CVs Scan rate: 0.1 V s -1 0 M DNA_2: M DNA_1: M i bi b i ai a 1 – i a /i b = Oxidized species, Fc +, was regenerated to be Fc by electron donor [Fe(CN) 6 ] 4-. Anodic current of Fc was amplified 1.75 times (= 0.362/0.207). Signal amplification with [Fe(CN) 6 ] 4- Without [Fe(CN) 6 ] 4- With [Fe(CN) 6 ] 4- e–e– Scheme3: Switching of Fc mediation Before hybridization After hybridization [Fe(CN) 6 ] 4- 22

23 Synthesis of the “signal-on” probe DTT NAP-5 1 CD-DNA-Fc, e–e– e–e– Fc hybridization electrode Detection system using probes without anchors enables detection in bulk solutions.

24 Conclusion 1. Upon hybridization, the inclusion complex between the terminal Fc and β-CD dissociates, restoring redox reaction of the Fc.  Label- and reagent-free, and “signal-on” detection system was developed. 2. Detection of target DNA - Redox potential: negative shift of Δ62 mV - Current change: 1.3 nA  6.9 nA (5-fold, at +0.3 V) e–e– e–e– Fc hybridization electrode A CD-DNA-Fc probe, based on a not only “self-reporting” but also “signal-on” architecture, leads us to a simple and rapid gene diagnosis. 24 Conclusion


Download ppt "Label-free and Reagent-free DNA Detection Based on Supramolecular Electrochemistry Hiroshi AOKI National Institute of Advanced Industrial Science and Technology."

Similar presentations


Ads by Google