WO2006134376A2 - Detection d'une capture chimique sans indicateur - Google Patents

Detection d'une capture chimique sans indicateur Download PDF

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Publication number
WO2006134376A2
WO2006134376A2 PCT/GB2006/002205 GB2006002205W WO2006134376A2 WO 2006134376 A2 WO2006134376 A2 WO 2006134376A2 GB 2006002205 W GB2006002205 W GB 2006002205W WO 2006134376 A2 WO2006134376 A2 WO 2006134376A2
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WIPO (PCT)
Prior art keywords
sers
chip
probe
molecules
calibration
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PCT/GB2006/002205
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English (en)
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WO2006134376A3 (fr
Inventor
Mino Green
Feng-Ming Liu
Peter Kollensperger
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Imperial Innovations Limited
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Publication of WO2006134376A2 publication Critical patent/WO2006134376A2/fr
Publication of WO2006134376A3 publication Critical patent/WO2006134376A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present disclosure relates to the detection of a target molecule by chemical capture (for example hybridization) between a probe and a target molecule using Surface Enhanced Raman Spectroscopy (SERS).
  • SERS Surface Enhanced Raman Spectroscopy
  • the target molecule is a bio-molecule. More particularly, the target molecule is an oligonucleotide.
  • SERS Surface Enhanced Raman Spectroscopy is a spectroscopic technique which combines laser spectroscopy with the optical properties of metallic nanostructures, resulting in strongly increased Raman signals when the studied molecules are attached to sub-micron sized gold or silver structures.
  • SERS is understood to include Surface Enhanced Resonant Raman Spectroscopy (SERRS).
  • SERRS Surface Enhanced Resonant Raman Spectroscopy
  • the molecules may be oligonucleotides.
  • the abovementioned discovery of well resolved SERS spectra for oligonucleotides has allowed the development of a method of detecting hybridisation based on the signature derived from the SERS spectra before and after hybridisation has occurred, thus providing a label or tag free method of detecting hybridization or other probe/target pairs.
  • the signatures may be derived from a set of predetermined peaks of the spectra using, for example, peak area or peak height.
  • the set of peaks may comprise two peaks and preferably a first peak is used which corresponds to the breathing mode of adenine and a second peak is used which corresponds to the breathing mode of thymine or cytosine.
  • the first peak may then be located at 731cm "1 and the second peak may be located at 790cm "1 .
  • the signatures are computed from the ratio of measures derived from the two peaks.
  • the method may include taking a calibration reading from a calibration region of the surface which is spatially distinct from the region used for detecting hybridisation.
  • the analysis can be calibrated for changes in the efficiency of the SERS substrate.
  • the calibration region supports a calibration molecule which preferably does not hybridise with the target molecules.
  • the calibration molecule may be a single strand oligonucleotide comprising exclusively adenine, thiamine, cytosine or guanine nucleotides.
  • the method may be advantageously applied to an analytical chip including a plurality of hybridisation (probe) regions distributed across the chip and preferably also including a plurality of calibration regions also distributed across the chip.
  • the invention further extends to analysis systems as defined in Claim 15, a computer program as defined in Claim 16 and a computer readable medium or data signal as defined in Claim 17.
  • a SERS chip including one or more regions of a SERS surface as defined in Claim 18. It can be shown that, (non-obviously), a SERS signal does not always increase with increase of target material when the concentration of probe molecules on the chip is increased. In particular, when the chip is covered with a dense packing of probe oligonucleotides, there may be insufficient space for the target molecules to insert themselves into this close packing. Accordingly, and unexpectedly, an improved signal can be obtained when the probe molecules on the chip are packed less densely than a close packing.
  • the packing of probe molecules is preferably substantially one half of the close packing, providing sufficient space for the target oligonucleotides while ensuring that the surface of the chip is at optimal coverage.
  • a SERS chip is provided as defined in Claim 21.
  • the probe molecules and/or the calibration molecules are preferably oligonucleotides.
  • a further aspect of the invention relates to the use of a SERS chip as defined in Claim 24.
  • a SERS chip as defined in Claim 24.
  • the number of probe molecules may exceed the number of target molecules such that the likelihood of a target molecule binding a probe molecule is maximised.
  • the converse is true and a number of probe molecules which is smaller than the number of target molecules may be more advantageous. This is because any probe molecule which does not hybridise when the sample is applied will result in an undesired background signal potentially affecting the results of the analysis adversely.
  • the number of probe molecules is sufficiently small with respect to the number of target molecules to ensure that substantially all the probe molecules are hybridized.
  • a SERS chip as defined above is used.
  • a SERS chip as defined in Claim 27.
  • the irregular array of metallic structures is thought to enhance the SERS characteristics of the substrate because, effectively, it provides a relatively wide sample of structure dimensions and geometry as compared to an array which is manufactured to be regular.
  • SERS is thought to be related to the shape and dimensions of the SERS surface, a random sample of structures is thought to be advantageous because it will ensure that at least a sub-range of the structures result in good enhancement.
  • a regular array of structures may produce a better signal if tuned exactly to the optimum parameters for SERS, but the optimum parameters are more likely to be missed completely for a regular spectra than for an irregular spectra sampling the space of structures and dimensions.
  • the surface includes silver structures supported on a silicon substrate and the structures may advantageously be torroids or pillars.
  • a method of applying probe/target molecules as claimed in Claim 32 is provided. Because the electrolyte in the buffer does not adsorb onto the SERS surface, the structural integrity of the surface is better maintained.
  • a reactive substrate as claimed in claim 34 is provided,
  • Figure 1 illustrates the manufacture of a SERS substrate
  • Figure 2 is a schematic depiction of a SERS substrate with attached oligonucleotide probes
  • Figure 3 shows a block diagram of a system implementing a method of detecting hybridisation
  • Figure 4 shows SERS spectra for two oligonucleotides before and after hybridisation
  • Figure 5 is a flow diagram of the method for detecting hybridisation
  • Figure 6 is a schematic depiction of a SERS substrate including calibration regions.
  • an oxide layer 2 typically 300 nm thick, is grown on a piece of silicon wafer.4
  • the oxidized silicon is then etched with H 2 O 2 (27.5 wt%) : NH 4 OH (0.88): water (1 : 1 : 1) to render it clean and hydrophilic.
  • a thin layer 6, thickness L (typically IOnm), of CsCl is evaporated on the clean surface.
  • the CsCl thin Film is then placed in an atmosphere of controlled relative humidity, whence it self-reorganizes into hemispherical islands 8.
  • the mean island diameter, ⁇ d>, and their coverage of the surface, F can be adjusted by the humidity, the original thickness, L, and development time.
  • the remnant Cr mask film is then stripped off, using a commercial Chrome Etchant (Rockwood Electronic Materials, UK).
  • This structure is then chemically etched to increase the average diameter of wells and to narrow the diameter distribution using an SiO 2 (not shown in Figure 1).
  • the substrates is washed in 69% HNO 3 , then NH 4 OH solution (7% ⁇ 8%NH 3 in water) for 2 min respectively, and then pre-etched in 1%HF solution for 1 min.
  • the substrate is transferred to a solution of 6mM AgNO 3 +0.5%HF for silver deposition: exposure is for 20s (no stirring), then 20s (60rpm stirring).
  • Silver deposition occurs on the patterned silicon by a process of galvanic exchange, viz:
  • Si + 4Ag + + 6F- SiF 6 2' + 4Ag
  • Silver structures 18 in the form of tori are formed at the bottom of the etched wells: a typical substrate used throughout the paper is as shown on Fig. 1.
  • the concentration of HF can be decreased to 0.12%, changing the dynamics of silver deposition to result in pillars of silver supported on a silicon substrate.
  • a suitable stabilization agent can be used (e.g. a surfactant) especially when very diluteDNA solutions ( ⁇ 10 '9 M) are used.
  • probes are deposited on the substrate. Immobilisation of oligonucleotide probes on a SERS surface can be achieved in any way known to a skilled person. Typically, 30 ⁇ l of stock 0.05mM of disulphide linked aqueous DNA solution (DNA-C 6 -S-S-C 6 -DNA) are mixed with lO ⁇ l of a solution that was 10 mM with respect to DTT in 0.1 M TEAA buffer; and kept for 2 h. The reducing agent, DTT, causes the rupture of the disulphide bonds, yielding DNA with the thiol group at the 3' end of the DNA strand, (HS-C 6 - DNA).
  • DTT disulphide linked aqueous DNA solution
  • the solutions are then applied to an Amersham G-25 MicroSpin column to remove the excess DTT and the percolate extracted twice with 700 ⁇ l ethyl acetate. Aliquots of this solution may then be diluted down with, for example, 0.1M TEAA to the desired concentration.
  • the functionalised oligonucleotides attach to the SERS structures via S-Ag bonds.
  • the solution of desired concentration is then applied to the SERS surface of the substrate using a commercially available micro printing device (a so called "mircro spotter”) for putting down drops of solution on a surface in specific areas.
  • the solution can be deposited in drops of a diameter down to 60 microns, theoretically allowing more than 10 4 different drops to be applied to a chip having a surface area of half a square centimetre.
  • the irregular array of structures obtained as described above has been found to result in SERS enhancements greater than known SERS substrates. Because SERS is thought to be related to the shape and dimensions of the SERS surface, a random sample of structures is thought to be advantageous because it will ensure that at least a sub-range of the structures result in good enhancement. By contrast, a regular array of structures may produce a better signal if tuned exactly to the optimum parameters for SERS, but the optimum parameters are more likely to be missed completely for a regular spectra than for an irregular spectra sampling the space of structures and dimensions.
  • the required concentration may be calculated using a suitable steric model or may be determined experimentally. In practice, the latter may be more effective and an optimal concentration of the probe solution can be determined by examining the overall intensity of the Raman spectrum, the intensity of certain features thereof (such as the intensity or area of one or more peaks), the ability to discriminate between the hybridised and non-hybridised state (see below) or a combination of the above as a function of probe concentration. Conveniently, this may be done efficiently by using a chip as described above to which drops of varying concentration are applied. The chip can then be scanned, as described below, before and after application of a solution containing target molecules and the results are analysed to select the probe concentration resulting in the best signal characteristics.
  • the supporting electrolyte does not adsorb onto the silver (or other metal) structures in order to avoid destabilising the structures.
  • F " ions do not adsorb onto the silver structures and a NaF buffer solution may be used.
  • the controller 26 controls a Raman spectrometer 28, for example, a Raman microscope and a sample handler 30.
  • the sample handler comprises a Raman cell accepting a chip to be analysed and, optionally, means for applying a sample containing target molecules to the chip.
  • the controller 26 controls the relative motion of the laser beam of the Raman spectrometer and the SERS surface of the chip inside the Raman cell. This can be achieved, for example, by deflecting the laser beam or by moving the chip on a translational stage, for example, by means of a piezo-electric manipulator.
  • the controller 26 further controls analyser 38 which includes electronic circuits, for example a micro computer, for analysing data obtained by the Raman spectrometer 28, as described in more detail below.
  • Figure 4 shows spectra obtained for oligonucleotide probes (lower traces) and probe-target hybridised complexes (upper traces) for bacterial DNA glnH and mammalian DNA OBP (thick and thin traces, respectively). The respective sequences are set out in the table below:
  • the ratio of the peak height of the peaks at 731 and 790cm "1 changes notably on hybridisation.
  • a signature of the spectrum as, for example, the ratio of the 731 cm “1 peak (corresponding to the breathing mode of cytosine and thymine) to the peak at 790cm "1 (corresponding to the breathing mode of adenine) one can detect the occurrence of hybridisation by a corresponding change in the signature. This forms the basis of the detection method described below. If the result obtained from these peaks is ambiguous, further peaks (either of the same or other nucleotides can be analysed analogously). Further details of SERS measurements and experimental results can be found in Green et al.
  • a chip functionised as described above, is placed in the Raman cell and is scanned to obtain a baseline spectrum for each region or dot of probe molecules at step 40. The obtained data is then used to calculate a baseline signature for each region of probe molecules at step at 42.
  • a sample is applied to the chip for a sufficient time to allow hybridisation to occur and the chip is washed at step 46. Both steps 44 and 46 can be carried out in any suitable way known to a skilled person and can either be integrated within the sample handler or be carried out off-line by removing the chip following the baseline scan and replacing it after step 46.
  • a sample scan is obtained analogously to the baseline scan at step 40.
  • a sample signature is calculated for each region at step 50.
  • the sample signature is compared to the baseline signature for each region to determine whether or not hybridisation has occurred in a given region. The identity of the target molecule will then be inferred from the identity of the probe molecule of regions where hybridisation occurred.
  • the baseline and sample signatures can be calculated as the ratio of the peaks (measured by peak height or peak area) of the breathing modes of adenine and cytosine/thymine. Other peaks may also be included in the analysis to resolve ambiguities or increase accuracy. However, other measures can be used to derive the signature, provided that a change between probe and probe/target complex molecules can be detected. Another possibility would be to regard the whole spectrum, suitably discretised, as the signature and detect a change of the signature by a suitable measure of similarity, for example, a dot product between spectra.
  • a SERS chip 20 including probe regions 54 and calibration regions 56 is depicted schematically in Figure 6.
  • the probe regions are distributed evenly across the chip and contain probe molecules attached to the surface of the chip, as described above. Interspersed with the probe regions, one or more calibration regions 56 are provided on the chip. These regions contain calibration molecules attached to the surface of the chip and can be used for calibration as described below.
  • a baseline scan 40 and a sample scan 48 are carried out in the same manner as described with reference to Figure 5, with the additional step of identifying the spectra obtained at each scan for the calibration regions. These calibration spectra are then used to calculate a normalisation factor which can be used to calibrate the measurement for any changes in overall SERS efficiency during application of the sample.
  • this calibration can be derived locally for probe regions adjacent to each calibration region, or a global normalisation factor can be calculated. In the latter case, a single suitably placed calibration region may be sufficient.
  • the normalisation factor may be derived in various ways, for example, using the peak intensity or peak area of a certain peak, and averaged over a number of peaks or the whole spectrum.
  • the calibrating molecule should be similar to the class of probe molecules used on the chip such that any change in the SERS efficiency of the substrate is comparable for the calibration molecule and the probe molecule.
  • it must be ensured that the calibration molecule does not hybridise with any of the target molecules corresponding to the probe molecules on the chip so that any changes in the calibration signal are attributed to changes in the SERS efficiency of the chip rather than hybridisation.
  • this can be conveniently achieved by using a calibration molecule consisting of an oligonucleotide having a sequence which does not (or is highly unlikely to) occur naturally.
  • an oligonucleotide sequence of only adenine, cytosine, guanine or thymine can be used.
  • an adenine only oligonucleotide is used because of its superior signal strength as compared to the other nucleotides.
  • the length of the calibration oligonucleotide can be matched to the length of probe oligonucleotides adjacent to the calibration region in question or can be chosen to correspond to the average sequence length of the probe nucleotides.
  • An alternative or additional approach to addressing the issue of deterioration of the chip with handling and exposure to sample solution is to stabilise the chip surface.
  • the above issue is overcome by coating the functionalized substrate with a thin layer of a water gel such as agarose or polyacrylamide.
  • a water gel such as agarose or polyacrylamide.
  • the gel material before the formation of the gel, is placed on the functionalized substrate (which has a hydrophilic surface) in the form of a thin liquid film, ca. lmm thick.
  • the properties of the gel typically 98% water are such as to allow virtually unimpeded passage of water, electrolytes, oligonuclitedes and small proteins, while at the same time retaining silver toroides in their initial position.
  • This stablization of a SERS surface using gel can be applied to structures other than those described in disclosure. It can be used anywhere there are structures that can be destabilized by chemical treatment and agitation. It is understood that the gel need not be water based and that for a hydrophobic surface a non- water based gel would be appropriate as long as it is lyophilic with the surface in question.
  • the two gel materials mentioned above are the most common materials used in bio-chemical laboratories, but there are many other gel materials that will prove suitable.
  • the requirements are passage of the requisite chemicals through a water medium.
  • the method of placing the gel on the surface can be simply by putting a drop of material on the surface, or placing material on the surface and spinning or draining excess material away or by placing material on the surface followed by photo-setting of a projected area of gel in the required place.
  • probe and target molecules are not oligonucleotides.
  • the probe/target molecules can be other bio-molecules such as antibodies/antigens for detecting the presence of certain proteins or any other molecule which can be hybridised or otherwise chemically captured with a probe molecule on the SERS chip such that a change in the corresponding Raman spectrum occurs.
  • the methods described above are applicable to detectors for only one target molecule. In this case, chip production can be simplified in that the probe molecules can be applied to the chip surface simply by submerging the chip in a suitable probe solution.
  • the method steps described above can be carried out in any suitable order.
  • the spectra resulting from baseline and probe scans can be stored on a suitable recording media and the calculation of the signatures and subsequent analysis can be carried out off line once all of the data is acquired.

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  • Bioinformatics & Cheminformatics (AREA)
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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne un procédé de détection sans étiquette d'une capture chimique utilisant un substrat SERS et des procédés d'obtention du substrat SERS convenablement fonctionnalisé.
PCT/GB2006/002205 2005-06-15 2006-06-15 Detection d'une capture chimique sans indicateur WO2006134376A2 (fr)

Applications Claiming Priority (2)

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GB0512230.4 2005-06-15
GB0512230A GB0512230D0 (en) 2005-06-15 2005-06-15 Hybridisation detection

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WO2006134376A2 true WO2006134376A2 (fr) 2006-12-21
WO2006134376A3 WO2006134376A3 (fr) 2007-06-21

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103030096A (zh) * 2011-10-09 2013-04-10 中国科学院高能物理研究所 一种具有纳米结构表面的硅材料及其制作方法
WO2013150290A1 (fr) 2012-04-05 2013-10-10 Renishaw Diagnostics Limited Procédé d'étalonnage d'appareil de spectroscopie équipement destiné à être utilisé dans le procédé
WO2015168205A1 (fr) * 2014-04-29 2015-11-05 University Of Houston System Procédé de spectrométrie de l'effet raman exalté de surface d'estampage pour détection et imagerie moléculaire, multiplexée, sans étiquette
CN111708269A (zh) * 2020-05-29 2020-09-25 张译文 一种表壳及其制造方法

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WO2005098441A2 (fr) * 2004-03-30 2005-10-20 Intel Corporation Procede de detection de liaison moleculaire par spectroscopie a effet raman superficiel exalte

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103030096A (zh) * 2011-10-09 2013-04-10 中国科学院高能物理研究所 一种具有纳米结构表面的硅材料及其制作方法
WO2013150290A1 (fr) 2012-04-05 2013-10-10 Renishaw Diagnostics Limited Procédé d'étalonnage d'appareil de spectroscopie équipement destiné à être utilisé dans le procédé
WO2015168205A1 (fr) * 2014-04-29 2015-11-05 University Of Houston System Procédé de spectrométrie de l'effet raman exalté de surface d'estampage pour détection et imagerie moléculaire, multiplexée, sans étiquette
US9546958B2 (en) 2014-04-29 2017-01-17 University Of Houston System Method of stamping surface-enhance Raman spectroscopy for label-free, multiplexed, molecular sensing and imaging
CN111708269A (zh) * 2020-05-29 2020-09-25 张译文 一种表壳及其制造方法

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WO2006134376A3 (fr) 2007-06-21

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