Method for concentrating analytes

Abstract

A method for concentrating at least one analyte including the following steps: preparing a first phase including at least one analyte; depositing a drop of the first phase on a substrate; depositing on the drop of first phase a drop of a second liquid phase including at least one surfactant, the second phase being non-miscible with the first phase; evaporating the drop of the second phase; and evaporating the drop of the first phase. Also relates to a method for detecting at least one analyte using the concentration method; and a system using the detection method.

Claims

1. A method for concentrating at least one analyte comprising the following steps: preparing a first phase comprising at least one analyte, wherein the first phase is an aqueous solution and does not comprise surfactant; depositing a drop of said first phase on a substrate; depositing on said drop of first phase a drop of a second liquid phase comprising 2-(Perfluorooctyl) ethyl alcohol as a non-volatile fluorinated surfactant, said second phase being a volatile oil; evaporating the drop of the second phase; and evaporating the drop of the first phase, wherein the deposited drop of the first phase has a deposition zone with a center, and after evaporation the at least one analyte is concentrated at the center of the deposition zone of the drop of the first phase.

2. The method for concentrating at least one analyte according to claim 1, wherein the volatile oil is a fluorinated.

3. The method for concentrating at least one analyte according to claim 1, wherein the steps of depositing a drop of said first phase and of depositing on said drop of first phase a drop of a second phase are successive.

4. The method for concentrating and detecting at least one analyte according to claim 1, wherein the substrate comprises a plurality of wells each of dimensions configured to receive a plurality of drops of the first phase and of the second phase.

5. A method for concentrating and detecting at least one analyte comprising: at least one step of concentrating at least one analyte using the concentration method according to claim 1; and detecting and/or analysing the at least one concentrated analyte by means of a physicochemical or biological analysis method.

6. The method for concentrating at least one analyte according to claim 2, wherein the volatile oil is a fluorinated oil of formula C.sub.nF.sub.2n+2 or C.sub.nF.sub.2n+1OC.sub.n′H.sub.2n′+1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 comprises a schematic representation of drop deposition according to the prior art, a deposition of two non-miscible drops and the deposition according to the present invention, and the images corresponding to said depositions after the evaporation of the second phase and of the first phase.

(2) FIG. 2 illustrates the influence of the method for concentrating at least one analyte on the detection of said analyte by MALDI-TOF mass spectrometry.

(3) FIG. 3 illustrates the influence of the method for concentrating at least one analyte on the linearity and reproducibility of the signal obtained by MALDI-TOF mass spectrometry.

(4) FIG. 4 illustrates the influence of the multi-deposition technique on the analysis plate according to the present invention on the detection of said analyte by MALDI-TOF mass spectrometry.

(5) FIG. 5 shows the MALDI-TOF analysis spectra of a peptide mixture concentrated as per the method according to the present invention.

(6) FIG. 6 illustrates the influence of the method for concentrating at least one analyte according to the present invention on the concentration of an analyte at the centre of the deposition zone.

(7) FIG. 7 represents a microfluidic device comprising 2 tubes or channels (5, 6), a junction 7 and a third tube or channel 8.

(8) FIG. 8 comprises a schematic representation of the deposition according to the present invention, and the image corresponding to said deposition after the evaporation of the second phase and the first phase.

EXAMPLES

(9) The present invention will be understood more clearly on reading the following examples illustrating the invention in a non-limiting manner.

Example 1: Enhancement of MALDI-TOF Mass Spectrometry Analysis Sensitivity by Concentrating the Analyte on a MALDI Plate

(10) Materials and Methods

(11) Materials

(12) a MALDI matrix solution (2 mg/ml α-cyano-4-hydroxycinnamic acid) comprising a mixture of acetonitrile and acidified distilled water in a 50:50 ratio; an oil containing 10% (m/m) 2-(Perfluorooctyl) ethyl alcohol; and a stock solution of angiotensin II, comprising the peptide at a concentration of 1 pmol/L in a solution of 0.1% trifluoroacetic acid.
Methods

(13) For each series of experiments, all the materials are prepared immediately prior to the analyses.

(14) The matrix solution is prepared by diluting the matrix to a concentration of 2 mg/ml in a mixture of acetonitrile and acidified distilled water in a 50:50 ratio. The mixture needs to be vortexed for 5 min, then placed for 10 min in an ultrasound bath for the solubilisation to be complete.

(15) The peptide stock solution is prepared by diluting angiotensin II to a concentration of 1 pmol/L in a solution of 0.1% trifluoroacetic acid and is stored at −20° C.

(16) Peptide aliquots at 100 fmol/μL are then prepared from an aliquot at 1 pmol/μL by diluting same in a distilled water/acetonitrile mixture (75:25). For each series of analyses, a fresh aliquot is diluted to the desired concentration with an acidified distilled water/acetonitrile mixture (75:25) containing the 2 mg/ml matrix.

(17) A first deposition is carried out conventionally, namely a drop of solution containing the peptides is deposited on a metal substrate using a pipette. An evaporation ensues, which will enable the non-volatile analytes to be trapped on the surface of the substrate.

(18) A second deposition is carried out by depositing successively a drop of solution containing the peptides and a drop of volatile oil free from surfactant on this first drop on a metal substrate using a pipette.

(19) A third deposition is carried out according to the method for concentrating at least one analyte as defined in the present invention.

(20) The first deposition and the third deposition are then analysed by MALDI-TOF-MS.

(21) Results

(22) The first two depositions described above induce a distribution of the analytes over the entire contact surface between the drop and the substrate, frequently resulting in coffee ring phenomena described above. The method for concentrating at least one analyte as defined in the present invention enables a concentration of said analyte at the centre of the surface occupied initially by the drop.

(23) FIG. 1 illustrates the three depositions carried out: the deposition according to the prior art comprising a drop of the first phase (1) containing at least one analyte (11) deposited on a substrate (2); the second deposition comprising a drop of the first phase (1) containing at least one analyte (11) topped with a drop of the second phase (3), free from surfactant, non-miscible with the first phase (1) deposited on a substrate (2); the third deposition on a substrate (2) according to the method for concentrating at least one analyte (11) as defined in the present invention comprising a drop of the first phase (1) containing at least one analyte (11) topped with a drop of the second phase (4) containing at least one surfactant (41), said second phase (4) being non-miscible with the first phase (1).

(24) In the mass spectra presented in FIG. 2, the signal intensity changes from 2085 to 60,000 for the conventional deposition and the deposition according to the method of the present invention, respectively, or an increase in the signal by a factor of 30.

Example 2: Deposition on MALDI Plate Using a Digital Microfluidic System

(25) Materials and Methods

(26) Materials

(27) a MALDI matrix solution (2 mg/ml α-cyano-4-hydroxycinnamic acid) comprising a mixture of acetonitrile and acidified distilled water in a 50:50 ratio; four solutions containing an analyte such as angiotensin II at different concentrations (from 1 nM to 20 nM); an oil containing 10% (m/m) 2-(Perfluorooctyl) ethyl alcohol.
Methods

(28) Four solutions containing angiotensin II at different concentrations (from 1 nM to 20 NM) were used separately to generate drops in a digital microfluidic system then deposited according to the concentration method according to the present invention on a commercial MALDI plate made of stainless steel for analysis. For each concentration, 10 repetitions were carried out, at the end whereof, the number of drops deposited and therefore the precise quantity of analyte deposited is calculated then correlated with the corresponding intensities obtained by the MALDI-TOF analysis.

(29) Results

(30) The limits of detection in terms of sensitivity of the apparatus are achieved according to the manufacturer documentation for the instrument used. The concentration method according to the present invention therefore makes it possible to detect very small quantities of peptide from a sub-nanomolar solution. Furthermore, a strong correlation between the intensity of the signal obtained with MALDI-TOF and the quantity of peptide deposited was observed (represented in FIG. 3), which confirms the reproducibility of the experiment, not achievable for a deposition method according to the prior art.

Example 3: Deposition on Plate Including Wells Using a Digital Microfluidic System

(31) Materials and Methods

(32) Materials

(33) a MALDI matrix solution (2 mg/ml α-cyano-4-hydroxycinnamic acid) comprising a mixture of acetonitrile and acidified distilled water in a 50:50 ratio; a solution of peptide, angiotensin II, at 0.1 nM; an oil containing 10% (m/m) 2-(Perfluorooctyl) ethyl alcohol; a silicon wafer; and a photosensitive resin.
Methods

(34) On a silicon wafer, previously cleaned, is deposited an SU-8 3035 type photosensitive wafer. The wafer is then placed in a spin-coater suitable for obtaining a uniform layer of resin on the silicon wafer. The wafer is then deposited on a heating plate for 30 minutes at 95° C. It is then placed in an ultraviolet exposure box and covered with a mask whereon is printed the shape of the wells. The wafer is then illuminated for 17 seconds at a power of 20 mW/cm.sup.2. The wafer is then deposited in a bath containing the development solution. After 10 minutes, the resin that has not been irradiated is dissolved and the wells appear. The wafer is cleaned with isopropanol then dried. The plate obtained comprises a plurality of wells 700 μm in diameter and 100 μm in height.

(35) These wells make it possible to collect the drops generated by the digital microfluidic system. A plurality of depositions according to the concentration method according to the present invention are carried out in the same well.

(36) Results

(37) By repeating the depositions of drops in the same well, the quantity of analytes present in this well is increased significantly. FIG. 4 compares a “single” deposition by the microfluidic system and a multi-deposition by the microfluidic system on the well plate, and the two depositions carried out according to the concentration method according to the present invention. The signal-to-noise ratio is enhanced and the intensity is increased by an order of magnitude.

Example 4: Deposition of a Peptide Mixture on MALDI Plate Using a Digital Microfluidic System

(38) Materials and Methods

(39) Materials

(40) a MALDI matrix solution (2 mg/ml α-cyano-4-hydroxycinnamic acid) comprising a mixture of acetonitrile and acidified distilled water in a 50:50 ratio; a solution containing an analyte such as a peptide mixture of concentration 2.5 nM; an oil containing 10% (m/m) 2-(Perfluorooctyl) ethyl alcohol.
Methods

(41) The deposition is carried out by forming drops, generated by a digital microfluidic system, of solution containing the peptide mixture then deposited according to the concentration method according to the present invention on a commercial MALDI plate made of stainless steel for analysis. An evaporation ensues, which will enable the non-volatile analytes to be trapped on the surface of the substrate.

(42) Results

(43) The MALDI-TOF analysis of such depositions enables the detection of 10 peptides from a deposited quantity of 250 attomoles of mixture. The gain of the present invention for the peptide mixture study is thus demonstrated given that the analysis involving a conventional deposition (drop of solution containing the peptides and drop of matrix solution are deposited on a metal substrate using a pipette) of a greater quantity of the same mixture (2500 atommoles) only allows the detection of 4 peptides.

(44) FIG. 5 shows the MALDI-TOF analysis spectra. The red markers correspond to the masses of the peptides detected.

Example 5: Deposition of a Protein on MALDI Plate

(45) Materials and Methods

(46) Materials

(47) a MALDI matrix solution (2 mg/ml α-cyano-4-hydroxycinnamic acid) comprising a mixture of acetonitrile and acidified distilled water in a 50:50 ratio; a solution containing an analyte such as a polypeptide chain (MMP12 protein labelled with a fluorophore: fluorescein); an oil containing 10% (m/m) 2-(Perfluorooctyl) ethyl alcohol.
Methods

(48) The deposition of a protein solution is carried out according to the concentration method according to the present invention. An evaporation ensues, which will enable the non-volatile analytes to be trapped on the surface of the substrate.

(49) Results

(50) The method for concentrating at least one analyte as defined in the present invention enables a concentration of a protein at the centre of the surface occupied initially by the drop, which is not observed in the case of a deposition by means of a conventional method (FIG. 6). Indeed, the deposition of the same protein solution by means of a conventional method (namely a drop of solution containing the protein is deposited on a metal substrate using a pipette) results in a distribution of the protein over the entire contact surface between the drop and the substrate, frequently leading to coffee ring phenomena described above.