Method and device for chemiluminescence-based analysis

Abstract

A method for detecting an analyte reactive towards luminol, comprising the steps of: feeding into a reaction chamber an alkaline solution of luminol, noble metal nanoparticles and at least one analyte reactive towards luminol, wherein the reaction chamber is in the form of a curved channel; detecting the light emitted due to a chemiluminescence reaction taking place in said channel; and discharging a reaction mass from said channel, characterized in that the average diameter of the metal nanoparticles is greater than 25 nm. Also provided is a microfluidic device for carrying out the method.

Claims

1. A method for detecting an analyte reactive towards luminol, comprising: feeding into a reaction chamber an alkaline solution of luminol, noble metal nanoparticles and at least one analyte reactive towards luminol, wherein the reaction chamber is in the form of a curved channel having serpentine-like shape, such that the form of the reaction chamber curves in alternate directions; detecting a light emitted due to a chemiluminescence reaction taking place in said curved channel; and discharging a reaction mass from said curved channel, wherein the average diameter of the noble metal nanoparticles is greater than 25 nm, wherein the curved channel comprises a plurality of straight sections including at least a first straight section, a second straight section, and a third straight section, which are parallel with each other, and wherein the straight sections are connected by a curved section, which joins the straight sections smoothly, creating the serpentine-like shape, and wherein a length L of each of the straight sections is from 400 μm to 1000 μm and a radius R of the curved section is from 50 μm to 200 μm, and wherein an enhancement of the light emitted by the chemiluminescence reaction in the second straight section is greater than an enhancement of the light emitted by the chemiluminescence reaction in the first and third straight sections.

2. A method according to claim 1, wherein the noble metal nanoparticles are selected from the group consisting of gold and silver.

3. A method according to claim 2, wherein the noble metal nanoparticles are silver.

4. A method according to claim 1, wherein the curved channel has cross-sectional dimension in the range from 0.15 mm to 0.5 mm.

5. A method according to claim 1, wherein reagents and reactants are fed into the reaction chamber at flow rates from 0.1 μL/sec to 0.5 μL/sec.

6. A method according to claim 5, wherein the flow rate is from 0.25 to 0.45 μL/sec.

7. A method for detecting an analyte reactive towards luminol, comprising: feeding into a reaction chamber an alkaline solution of luminol, noble metal nanoparticles and at least one analyte reactive towards luminol, wherein the reaction chamber is in the form of a curved channel having serpentine-like shape, wherein the curved channel is not spiral-shaped; detecting a light emitted due to a chemiluminescence reaction taking place in said curved channel; and discharging a reaction mass from said curved channel, wherein the curved channel consists of a plurality of straight sections including a first straight section, a second straight section, and a third straight section, which are parallel with each other, connected by a curved section, which joins the straight sections smoothly, creating the serpentine-like shape, and wherein a length L of each of the straight sections is from 400 μm to 1000 μm and a radius R of the curved section is from 50 μm to 200 μm, and wherein an enhancement of the light emitted by the chemiluminescence reaction in the second straight section is greater than an enhancement of the light emitted by the chemiluminescence reaction in the first and third straight sections.

8. A microfluidic device adapted for luminescence-based detection, comprising: A) a curved channel which has serpentine-like shape, having cross-sectional dimension from 0.15 mm to 0.5 mm; wherein the curved channel is not spiral-shaped, and wherein the flow channel consists of a plurality of straight sections including a first straight section, a second straight section, and a third straight section, essentially parallel sections connected by a curved section joining the straight sections smoothly, creating the serpentine-like shape, and wherein the length L of each of the individual straight sections is from 400 μm to 1000 μm and the radius R of the curved section is from 50 μm to 200 μm, and wherein an enhancement of the light emitted by the chemiluminescence reaction in the second straight section is greater than an enhancement of the light emitted by the chemiluminescence reaction in the first and third straight sections; B) a plurality of reservoirs and pumps for holding and delivering into said flow channel: a solution of a luminescence reagent, a luminescence enhancer comprising noble metal nanoparticles; a sample comprising an analyte reactive towards luminol; wherein said reservoirs are connected through tubes to input opening(s) of the flow channel, C) a detector for measuring the intensity of the light emitted by the luminescence reaction; and optionally D) a vessel to which the reaction mixture is withdrawn.

9. A microfluidic device according to claim 8, wherein the curved channel is fabricated in poly(dimethylsiloxane).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1a is a photograph of a microfluidic device comprising a serpentine-like channel, syringes for feeding reagents and a charge-coupled-device detecting emitted light.

(2) FIGS. 1b and 1c illustrate noble metal nanoparticles (1b) that were added to the serpentine-like channel (1c).

(3) FIG. 2a shows the reaction chamber of Example 6 (with gold nanoparticles) on the left side, and the reaction chamber of Reference Example 1 (without additives) on the right side.

(4) FIG. 2b is a plot of the luminescence intensity versus position along the flow path (serpentine arm).

(5) FIG. 2c is an illustration of a part of the serpentine-like channel, consisting of two straight, essentially parallel sections joined by a curved section.

(6) FIGS. 3a and 3b are plots of luminescence intensity versus serpentine arm for gold (3a) and silver (3b) nanoparticles.

EXAMPLES

Example 1 (Reference), 2-5 (Comparative) and 6-7 (of the Invention)

(7) The microluidic device used in the experiment comprises a serpentine-like channel formed in PDMS by the technique described above. We have designed and fabricated a reusable microflow device with a serpentine channel 200 μm in width, 200 μm in depth and 600 μm in length (for a single straight chain). The PDMS channel was molded over the 3D printed device. The layout for the mold was designed using the CAD Autodesk inventor (Stockport, UK). After printing, the channels were sealed using oxygen plasma for 30 s.

(8) An illustrative microfluidic device is shown in FIG. 1. In the one used in the experiment, the so-formed channel (1) consisted of eight straight parallel sections. Each pair of adjacent sections is connected by a toroidal section (2) of small radius which joins the straight sections smoothly, thereby creating a flow path with a serpentine-like shape. The straight sections are designated herein by Roman numbers I to VIII. The opening (3) of section I functions as a port for feeding the reagents. A first syringe (4) is used to inject an alkaline solution comprising luminol and the tested additive. A second syringe (5) is used to inject an aqueous solution of the agent reactive towards luminol. The diameter of the syringes is 180 μm. The outlet openings of the two syringes are connected to the opening (3) via 0.2 mm tubes (6) and (7), respectively. The reaction products are discharged through the open end of the last straight section and are collected in to vessel (8). The intensity of emitted light was detected (9) by a charge-coupled-device (CCD), Lumenera Infinity 2-3C (from Lumenera Corporation, Canada).

(9) The additives tested are gold and silver nanoparticles, available commercially from BBI or Sigma Aldrich in the form of suspensions in water stabilized with polyethylene glycol. Suspensions of monodisperse metal nanoparticles are available in various sizes. The properties of the additives tested are set out in Table 1 (information according to manufacturer):

(10) TABLE-US-00001 TABLE 1 Particles radius (nm) Concentration (number per ml) Gold 10 7 × 10.sup.11 nanoparticles 20 9 × 10.sup.10 30 2.6 × 10.sup.10   Silver 10 7 × 10.sup.10 nanoparticles 20 9 × 10.sup.9  30 2.6 × 10.sup.9   

(11) The Solutions used in the experiments are tabulated below:

(12) TABLE-US-00002 TABLE 2 Solution Ex. Reagent in syringe 1 in syringe 2 Reference Example 1 0.4 g luminol dissolved in an alkaline solution 50 mg of NaOCl (4 g NaOH dissolved in 1950 ml water) dissolved in 1950 ml water Comparative Examples 2 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved in 1950 ml water) dissolved in Additive: 50 ml gold nanoparticles suspension 1950 ml water (r = 10 nm) 3 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved in 1950 ml water) dissolved in Additive: 50 ml gold nanoparticles suspension 1950 ml water (r = 20 nm) 4 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved in 1950 ml water) dissolved in Additive: 50 ml silver nanoparticles suspension 1950 ml water (r = 10 nm) 5 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved in 1950 ml water) dissolved in Additive: 50 ml silver nanoparticles suspension 1950 ml water (r = 20 nm) Examples of the Invention 6 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved dissolved in ml water) dissolved in Additive: 50 ml gold nanoparticles suspension 1950 ml water (r = 30 nm) 7 0.2 g luminol dissolved in an alkaline solution 50 mg of NaOCl (2 g NaOH dissolved in ml water) dissolved in Additive: 50 ml silver nanoparticles suspension 1950 ml water (r = 30 nm)

(13) Several experiments were conducted at different flow rates of the reagents, in the range from 0.1 μL/sec to 0.5 μL/sec. The results reported below correspond to the experiments where the flow rate was 0.35 μL/sec, seeing that the maximal chemiluminescence intensity was obtained at said flow rate.

(14) The limit of detection for the experimental setup was determined using 3 standard deviations and 20 repeats of images for each individual point, and was estimated to be less than 110 μg/mL.

(15) In general, the experimental results captured by the CCD camera show a glowing serpentine channel for Examples 2 to 7, as opposed to a barely seen serpentine channel in the case of Reference Example 1 (devoid of any additive). Illustrative images corresponding to Example 6 (gold nanoparticles with radii of 30 nm used as chemiluminescence enhancer) vis-à-vis Reference Example 1 are provided in FIG. 2a—left and right images, respectively.

(16) Aside from images taken by a CCD camera, the results can also be presented graphically by plotting the intensity of the chemiluminescence emission of luminol as a function of the position along the flow path. That is, the intensity is measured for each of the straight sections of the serpentine-like channel—serpentine arms I-VIII. The results are presented in FIGS. 3a and 3b, for the additives consisting of gold nanoparticles and silver nanoparticles, respectively: FIG. 3a corresponds to Examples 2, 3 and 6 and FIG. 3b corresponds to Examples 4, 5 and 7. The results for the Reference Example 1 are included in both graphs, and are indicated by rhombuses. The following observations can be made:

(17) (i) The results indicate that the addition of either gold nanoparticles or silver nanoparticles produces strong enhancement of the chemiluminescence intensity of luminol. For both metals, nanospheres with average size of 10 nm and 20 nm produce essentially the same effect. But further increase of the particle size of the metal additive to 30 nm leads to a noticeable increase in the enhancement of the chemiluminescence intensity of luminol.

(18) (ii) Taking into consideration the different concentrations of silver and gold in the commercially available suspensions used (see Table 1), one may conclude that silver nanoparticles induce a stronger enhancement of chemiluminescence than gold nanoparticles. The enhancement of luminol emission using silver nanospheres is stronger by a factor of up to 90 compared to using the same concentration of gold nanospheres.

(19) (iii) The results also illustrate the change in the intensity of emission with the distance along the serpentine channel, seeing that the strongest enhancement occurs in arm II, which can be understood to indicate the best mixing between reagents in this arm and/or the most favorable distance between the light emitting species and “nanoantennas”. The mixing in the microfluidic device occurs based on the diffusion of particles from one laminar layer into the adjacent one. Efficient mixing occurs around the bends due to the Dean flow; therefore, arm II after the first bend shows the highest chemiluminescence intensity.