Methods and Systems for Aldehyde Detection
Methods, systems and reagents are provided for detecting and quantifying carbonyl containing moieties in a variety of sample types.
1 . A method for detecting the presence of at least one aldehyde in a sample, the method comprising the steps of:
(a) dispensing reactive labeling reagent in solution on to a capture column such that the capture column is pre-loaded with reactive labeling reagent;
(b) pushing the sample through the column to form a labeled sample, wherein the labeled sample comprises one or more labeled compounds;
(c) eluting the one or more labeled compounds from the capture column with an organic solvent solution selected from DMF, DMSO, ethanol, acetonitrile, water miscible solvent, n-methyl pyrrolidone, and methanol/water/HCl solution;
(d) dispensing the one or more labeled compounds onto a separation column;
(e) separating the one or more labeled compounds from one another by using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer; and
(f) detecting the separated one or more labeled compounds in the solution, wherein the separated one or more compounds is identified as being at least one aldehyde.
2 . The method of claim 1 , wherein step (a) further comprises drying the capture column after being pre-loaded with reactive labeling reagent.
3 . The method of claim 1 , further comprising (g) measuring the concentration of the at least one aldehyde.
4 . The method of claim 1 , wherein the time elapsed from (b) through (f) is less than about 1 hour.
5 . The method of claim 1 , wherein the time elapsed from (b) through (c) less than about 10 minutes.
6 . The method of claim 1 , wherein the at least one aldehyde is selected from the group consisting of a C1 aldehyde, a C2 aldehyde, C3 aldehyde, a C4 aldehyde, a C5 aldehyde, a C6 aldehyde, a C7 aldehyde, a C8 aldehyde, a C9 aldehyde, a C10 aldehyde, and mixtures thereof.
7 . The method of claim 1 , wherein the at least one aldehyde is aliphatic, a di-aldehyde, or an aromatic aldehyde, or mixtures thereof.
8 . The method of claim 1 , wherein the sample comprises two or more aldehydes of different carbon chain lengths, and wherein the step of detecting the labeled aldehyde resolves each aldehyde.
9 . The method of claim 1 , wherein the sample is a biological sample.
10 . The method of claim 1 , wherein the sample is an environmental sample.
11 . The method of claim 1 , wherein the sample is selected from the group consisting of a breath sample, a urine sample, a blood sample, a plasma sample, and a sample of the headspace in a culture.
12 . The method of claim 1 , wherein the sample is a breath sample.
13 . The method of claim 1 , wherein the capture column comprises a material selected from the group consisting of glass, silica, polyethylene, Teflon, x9908, por-4903, polypropylene, and mixtures thereof.
14 . The method of claim 1 , wherein the capture column comprises acid activated silica.
15 . The method of claim 1 , wherein the methanol/water/HCl elution solution comprises HCl in an amount of about 0.1% to about 2.0% by volume.
16 . The method of claim 1 , wherein the reactive labeling agent comprises a fluorophore, a linker, and a reactive group.
17 . The method of claim 16 , wherein the fluorophore is selected from the group consisting of tetramethyl rhodamine (TAMRA), 5(6) TAMRA, 5 TAMRA, 6 TAMRA, rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110 (R110), and a coumarin.
18 . The method of claim 16 , wherein the linker is selected from the group consisting of substituted alkyl-diamines (C2-C10), substituted amino-carboxylic acids (C2-C10), and substituted polyethylene glycols (N=1-10).
19 . The method of claim 16 , wherein the linker is selected from the group consisting of hexanoic acid, aminohexanoic acid, cadavarine, polyethylene glycol, and polyglycol.
20 . The method of claim 16 , wherein the reactive group is selected from the group consisting of a hydrazine moiety, a carbohydrazide moiety, a hydroxylamine moiety, a semi-carbazide moiety, an aminooxy moiety, and a hydrazide moiety.
21 . The method of claim 1 , wherein the reactive labeling agent is selected from the group consisting of
and mixtures thereof.
22 . The method of claim 1 , wherein the step of detecting the aldehyde comprises measuring fluorescence emission produced by excitation of the fluorophore.
23 . The method of claim 1 , wherein the step of detecting the aldehyde comprises measuring fluorescence absorbance produced by excitation of the fluorophore.
24 . The method of claim 1 , wherein the step of detecting the aldehyde comprises directing light within a predetermined wavelength range to the labeled aldehyde, thereby producing a fluorescence, and detecting the fluorescence.
25 . The method of claim 3 , wherein the concentration of the aldehyde is determined by calculating the fluorescence excitation or emission relative to a standard curve, wherein the fluorescence signal is proportional to the concentration of the aldehyde.
26 . A method for detecting the presence of at least one carbonyl containing moiety in a sample, the method comprising the steps of:
dispensing reactive labeling reagent in solution onto a capture column,
pushing the sample through the column,
eluting labeled sample with an organic solvent solution,
dispensing labeled sample onto a separation column,
separating the labeled carbonyl containing moiety using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer, and
detecting the labeled carbonyl containing moiety,
wherein the method of detecting is complete in less than about 1 hour.
27 . The method of claim 26 , wherein the carbonyl containing moiety is selected from the group consisting of aldehydes, ketones, carboxylic acids and mixtures thereof.
28 . A method of detecting carbonyl containing moieties in a gas sample, the method comprising:
dispensing reactive labeling reagent in solution onto a capture column,
pushing the sample through the column,
eluting labeled carbonyl containing moieties with an organic solvent solution, dispensing labeled sample onto a separation column,
separating the labeled aldehydes using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer, and
exciting the labeled carbonyl containing moieties exiting the column, and
detecting the carbonyl containing moieties by measuring the fluorescence emitted from or absorbed by the labeled carbonyl containing moieties,
wherein the step of detecting resolves the carbonyl containing moieties based on the carbon chain length of the individual carbonyl containing moieties, and
wherein the time elapsed from pushing the sample through the column to detecting the carbonyl containing moieties is less than about 1 hour.
29 . A system for detecting the presence of at least one carbonyl containing moiety in a sample, the system comprising:
a sample capture container,
a device comprising a capture column, wherein a labeling reagent is deposited on the capture column, a separation column, elution solutions, a pump, a light for inducing fluorescence, a detection chamber, and a detector for measuring fluorescence emission, excitation, or absorbance of at least one labeled carbonyl containing moiety.
30 . The system of claim 29 , wherein the device receives a sample containing at least one carbonyl containing moiety from the sample capture container, deposits the sample onto the capture column embedded with the labeling reagent, performs an elution process on the column to elute the labeled carbonyl containing moiety, dispenses the labeled carbonyl containing moiety onto a separation column, separates the labeled carbonyl containing moiety exiting the column, measures the labeled carbonyl containing moiety, and presents data identifying the at least once carbonyl containing moiety.
31 . The system of claim 29 , further comprising one or more standards for measuring the concentration of the at least one carbonyl containing moiety.
32 . A method for detecting the presence of at least one carbonyl containing moiety in a sample, the method comprising the steps of:
(a) dispensing buffer in solution on to a solid substrate and drying the substrate;
(b) dispensing catalyst in solution on to a solid substrate and drying the substrate;
(c) dispensing reactive labeling reagent in solution on to a solid substrate and drying the substrate;
(d) layering a solid capture substrate, the substrate from (a), the substrate from (b), and the substrate from (c) in series;
(e) pushing the sample through the column, where the at least one carbonyl containing moiety is captured by the solid capture substrate;
(f) eluting the carbonyl containing moiety with an organic solvent solution selected from DMF, DMSO, ethanol, acetonitrile, water miscible solvent, n-methyl pyrrolidone, and methanol/water/HCl solution, whereby the carbonyl containing moiety, the buffer, the catalyst, and the reactive labeling reagent form an elution/reaction solution;
(g) incubating the elution/reaction solution to obtain at least one labeled carbonyl containing moiety;
(h) dispensing the solution containing at least one labeled carbonyl containing moiety onto a separation column;
(i) separating the labeled carbonyl containing moiety using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer; and
(j) detecting at least one labeled carbonyl containing moiety in the solution.
33 . A method for detecting the presence of at least one carbonyl containing moiety in a sample, the method comprising the steps of:
(a) dispensing reactive labeling reagent in solution on to a capture column and drying the column;
(b) pushing the sample through the column;
(c) eluting labeled sample with an organic solvent solution selected from DMF, DMSO, ethanol, acetonitrile, water miscible solvent, n-methyl pyrrolidone, and methanol/water/HCl solution;
(d) dispensing labeled sample onto a separation column;
(e) separating the labeled carbonyl containing moiety using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer; and
(f) detecting at least one labeled carbonyl containing moiety in the solution.
34 . A method for detecting the presence of at least one carbonyl containing moiety in a sample, the method comprising the steps of:
(a) dispensing buffer in solution on to a solid substrate and drying the substrate;
(b) dispensing catalyst in solution on to a solid substrate and drying the substrate;
(c) dispensing reactive labeling reagent in solution on to a solid substrate and drying the substrate;
wherein one or more of the buffer, the catalyst, and the reactive labeling agent are dispensed onto the same solid substrate, and layering a solid capture substrate in series with the substrates of (a), (b), and (c);
(d) pushing the sample through the column, where the at least one carbonyl containing moiety is captured by the solid capture substrate;
(e) eluting the carbonyl containing moiety with an organic solvent solution selected from DMF, DMSO, ethanol, acetonitrile, water miscible solvent, n-methyl pyrrolidone, and methanol/water/HCl solution, whereby the carbonyl containing moiety, the buffer, the catalyst, and the reactive labeling reagent form an elution/reaction solution;
(f) incubating the elution/reaction solution to obtain at least one labeled carbonyl containing moiety;
(g) dispensing the solution containing at least one labeled carbonyl containing moiety onto a separation column;
(h) separating the labeled carbonyl containing moiety using isocratic methods or changing gradients (linear, stepwise, piecewise, or parabolic), using methanol or water miscible solvent and water and/or buffer; and
(i) detecting at least one labeled carbonyl containing moiety in the solution.
35 . A column comprising a solid substrate and a reactive labeling reagent embedded onto the substrate.