Granted Patent B2

US 12,270,821 · App. 17/204,158 · Granted Apr 8, 2025

Inventors: Vishal Verma (Savoy, IL); Haoran Yu (Urbana, IL); Joseph Puthussery (Champaign, IL)

Assignee: The Board of Trustees of the University of Illinois

G01N35/1016B01L3/502B01L3/567B01L7/00G01N21/31G01N21/6428B01L2200/026B01L2200/0605B01L2300/18B01L2400/0403B01L2400/0622G01N2021/6439

Abstract

Claims (77)

1. An instrument for analyzing oxidative potential of particles from an air sample comprising:

a sample injector comprising:

a plurality of reservoirs including at least one reaction vial (RV) and at least one measurement vial (MV), each reservoir configured to hold at least one material selected from the group consisting of: a reaction chemical, an incubation chemical, a cleaning liquid, air, and waste fluid;

a plurality of automated programmable pumps for flowing the material from each reservoir to another reservoir or to or from an automated measurement system;

an automated multi-position valve and fluid conduits to fluidically connect a sample vial to the plurality of reservoirs, wherein the programmable pump(s) are configured to drive a flow of fluid sample from the sample vial to the reaction vial;

an automated valve actuator for controlling the multi-position valve to select different samples for analysis and fluidically control fluid flow within, into and out of the sample injector;

an automated sample incubator comprising:

an automated thermal mixer fluidically connected to the sample injector for automatically incubating and mixing reaction mixtures: wherein the sample incubator is configured to prepare each reaction mixture that comprises the fluid sample having said particles from the air sample and a material from the plurality of reservoirs, and wherein each reaction mixture is contained in the RV;

wherein said particles are PM 2.5 airborne particles having an effective diameter that is less than 2.5 μm;

wherein the automated sample incubator is configured to prepare at least a first reaction mixture comprising a first portion of the particles and dithiothreitol (DTT), a second reaction mixture comprising a second portion of the particles, surrogate lung fluid (SLF), and disodium terephthalate (TPT), and a third reaction mixture comprising a third portion of the particles and SLF;

the automated measurement system fluidically connected to the sample incubator and/or sample injector comprising:

a spectrophotometer configured to detect an optical absorbance of a first targeted compound for determining a consumption rate of ascorbic acid and an optical absorbance of a second targeted compound for determining a consumption rate of dithiothreitol;

wherein the measurement system is configured to detect the optical absorbances at least at a first absorbance wavelength and a second absorbance wavelength different from the first absorbance wavelength;

wherein the instrument is configured to automatically provide a portion of the first reaction mixture and a portion of the third reaction mixture to the spectrophotometer configured to automatically detect the optical absorbances of the first targeted compound and of the second targeted compound;

a spectrofluorometer configured to measure a fluorescence of a first indicator compound for determining a consumption rate of glutathione and a fluorescence of a second indicator compound for determining a generation rate of hydroxyl radicals;

wherein the measurement system is configured to measure the fluorescence at least at a first fluorescence wavelength different from each of the first and second absorbance wavelengths;

wherein the instrument is configured to automatically provide portions of each of the first, second, and third reaction mixtures to the spectrofluorometer configured to automatically measure the fluorescence of the first indicator compound in the first and second reaction mixtures and of the second indicator compound in the third reaction mixture;

an automated analyzer configured to calculate and output the consumption rate of ascorbic acid and the consumption rate of dithiothreitol using the absorbance of the targeted compounds measured by the spectrophotometer and configured to calculate and output the consumption rate of glutathione and the generation rate of hydroxyl radicals using the fluorescence of the indicator compounds measured by the spectrofluorometer, and thereby analyze the oxidative potential of the particles via the calculated and output endpoints comprising:

consumption rate of ascorbic acid (AA) in surrogate lung fluid (SLF) (OP AA-SLF );

consumption rate of glutathione (GSH) in SLF (OP GSH-SLF );

generation rate of hydroxyl radicals (·OH) in SLF (OP OH-SLF );

consumption rate of dithiothreitol (DTT) (OP DTT ); and

generation rate of hydroxyl radicals in DTT (OP OH-DTT ); and

an automated controller for automatically controlling fluidics with a control scheme;

wherein the controller, sample injector, sample incubator, measurement system, and analyzer are configured to provide an automated output of the endpoints in an output time that is less than 3 hours after the instrument receives the particles from the air sample.

2. The instrument of claim 1 , wherein the instrument is configured to determine the:

OP AA-SLF , OP GSH-SLF , and OP OH-SLF from a first stage SLF protocol; and

OP DTT and OP OH-DTT from a second stage dithiothreitol (DTT) protocol.

3. The instrument of claim 1 , wherein the instrument is configured to simultaneously determine a plurality of the endpoints.

4. The instrument of claim 1 , wherein the particles comprise atmospheric particulate matter having a diameter less than or equal to 2.5 μm extracted in a liquid solution.

5. The instrument of claim 1 , wherein the automated measurement system is configured to simultaneously detect an optical absorbance using the spectrophotometer and measure a fluorescence using the spectrofluorometer.

6. The instrument of claim 1 , wherein the material in the plurality of reservoirs comprises one or more of: K-PB (potassium phosphate buffer) of pH=7.4; SLF (surrogate lung fluid); TPT (Disodium terephthalate); DI (de-ionized water); OPA (O-pthaldialdehyde); DTT (dithiothreitol); or DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)).

7. The instrument of claim 6 , comprising three programmable pumps, wherein:

a first pump positioned in fluidic communication with DTNB, K-PB, SLF, DTT, reaction vials 1, 2 and 3, and measurement vials 1 and 2;

a second pump positioned in fluidic communication with reaction vials 1, 2 and 3, measurement vial 1, a spectrophotometer capillary cell, and the multi-position valve; and

a third pump positioned in fluidic communication with the OPA, TPT, spectroflourometer flow cell, reaction vials 1, 2 and 3, and measurement vial 1.

8. The instrument of any of claim 1 , wherein the instrument is configured to automatically and periodically determine each of the five endpoints over a time course of between 30 minutes and 90 minutes.

9. An automated method for analyzing oxidative potential of particles from an air sample, the method comprising the steps of:

(a) fluidically connecting a liquid sample vial containing particles extracted from the air sample to the sample injector system of claim 1 ;

(b) determining the OP AA-SLF , OP GSH-SLF , and OP OH-SLF SLF-based endpoints by:

(i) introducing with the sample injector a first sample volume from the liquid sample vial to a first reaction vial and a second sample volume to a second reaction vial, wherein the reaction vials each contain a buffer and SLF, and the second reaction vial further contains TPT to immediately capture hydroxyl radicals generated in a reaction in the reaction vial;

(ii) incubating the first and second RVs for an SLF incubation time period;

(iii) transferring a first aliquot from the first RV to a first MV and a second aliquot from the first RV to a second MV, wherein the transferring is by the sample injector;

(iv) diluting the first MV with DI for measuring AA;

(v) adding OPA into the second MV for probing GSH, wherein GSH reacts with OPA to form the indicator compound, and the indicator compound is delivered to the spectrofluorometer for indicator compound detection;

(vi) flowing the diluted sample from the first MV to a capillary cell of the spectrophotometer to measure absorbance of the targeted compound to determine a concentration of residual AA;

(vii) further diluting the sample in the second MV and injecting the diluted sample into a flow cell of the spectrofluorometer to measure fluorescence intensity to determine a concentration of GSH;

(viii) cleaning the RVs, MVs, flow cell and capillary cell with DI by the sample injector after each measuring step; and

(ix) repeating steps (ii)-(vi) over a time course to determine consumption rates of AA and GSH; and

(x) quantifying AA and GSH from calibration curves of initial absorbance and fluorescence intensity of different known concentrations of AA and GSH in SLF to thereby determine OP AA-SLF and OP GSH-SLF ;

(c) wherein measurement of OH is conducted at substantially the same time as GSH and AA measurement, the method further comprising the steps of:

(xi) reacting TPT with the ·OH in the second RV to form a fluorescent product 2-OHTA;

(xii) periodically collecting at various time intervals an aliquot from the second RV containing 2-OHTA;

(xiii) diluting the collected aliquot with DI;

(xiv) providing the diluted aliquot to a flow cell of the spectroflourometer to determine a concentration of the 2-OHTA to thereby determine OP OH-SLF ;

(d) wherein the flow cell and RV are cleaned after each determining step.

10. The method of claim 9 , wherein sample in the liquid sample vial containing particles from the air sample is prepared by the steps of:

collecting ambient PM 2.5 samples on a filter;

extracting the collected PM 2.5 from the filter; and

suspending the extracted PM 2.5 in liquid.

11. The method of claim 9 , wherein the simultaneous measurement of endpoints results in an elapsed measurement time to obtain all five endpoints that is less than or equal to three hours for a given ambient PM aqueous extract.

12. The method of claim 9 , further comprising the step of automatically controlling the multi-position valve and programmable pumps to provide desired flow-rates, fluid volumes, fluid composition, to and between vials and the measurement system.

13. The method of claim 12 , wherein the controlling step is by implementing a control scheme with a controller operably connected to the programmable pumps and the valve actuator.

14. The method of claim 13 , wherein the controller is embedded in a computing device.

15. The method of claim 9 , wherein after completing all determinations of AA and GSH in the first reaction vial and ·OH in the second reaction vial, a final cleaning step comprising rinsing with DI all the reaction vials, measurement vials, fluidic components and flow cells.

16. The method of claim 15 , wherein after the final cleaning step, the method further comprises determining the OP DTT and OP OH-DTT DTT-based endpoints, wherein the OP DTT determination comprises the steps of:

introducing with the sample injector a first sample volume to the first reaction vial, wherein the first RV contains DTT, and a K-PB-buffered TPT;

incubating the first RV for a DTT incubation time period;

periodically transferring an aliquot from the first RV with the sample injector to the first measurement vial containing DTNB;

forming 2-nitro-5-thiobenzoic acid (TNB) from the DTNB and residual DTT in the first MV;

diluting the TNB;

flowing the diluted TNB from the first MV to a capillary cell of the spectrophotometer to measure an absorbance of the TNB to determine a concentration of TNB and thereby OP DTT ; and

simultaneously to the OP DTT determination steps, the OP OH-DTT determination comprises the steps of:

periodically transferring an aliquot from the first RV with the sample injector to the second measurement vial at various time intervals;

diluting the aliquot in the second MV with DI;

delivering the diluted aliquot to a flow cell of the spectrofluorometer to determine a concentration of the 2-OHTA to thereby determine OP OH-DTT .

17. The method of claim 16 , wherein after completing all determinations of DTT and ·OH, the method further comprises a rinsing with DI all the reaction vials, measurement vials, fluidic components and flow cells.

Assignments (2)

Continuity (2)

References Cited (82)