WO2010053342A1

WO2010053342A1 - System and method for collecting and reading of fluorescence in capillary tubes

Info

Publication number: WO2010053342A1
Application number: PCT/MX2008/000152
Authority: WO
Inventors: Alberto MORALES VILLAGRÁN
Original assignee: Morales Villagran Alberto
Priority date: 2008-11-05
Filing date: 2008-11-05
Publication date: 2010-05-14

Abstract

A system and method is described for collecting and reading of fluorescence for the quantitative analysis of different substances through the use of enzymatic biosensors and reactors. The system comprises the collection of samples in sealed capillary tubes which are placed in a carrying device, there being a specific volume of the samples to be analysed together with a similar volume of enzymatic reactor or chromophore, wherein the fluorescence may be measured by a system of fluorescence detection, excitation and emission light being carried by optical fibres.

Description

SISTEMAY METHOD FOR COLLECTING AND READING FLUORESCENCE IN CAPILLARS

BACKGROUND OF THE INVENTION

Field of the Invention.

The present invention relates to fluorescence reading systems for quantitative analysis of substances, and more specifically it relates to a system for collecting and reading fluorescence in capillaries for the identification and quantification of substances through the use of enzymatic biosensors and reactors.

Related prior art.

In order to make the analysis more efficient and repetitively, a series of automatic fluorescence, optical density or luminescence reading systems (plate readers or "piate readers") have been manufactured. These systems are equipped with fluorescent detectors and in some cases with automatic reagent dosing systems for the induction of fluorescence or chemiluminescence. However, the minimum amount of sample to be used is generally 200 microliters and with the ability to automatically read 96 or more samples, depending on the type of equipment. Some fluorescence detectors have been modified for the reading in small samples, in which the cell has been adapted to measure in a volume of some 20 microliters, although the taking of each reading is done manually. In this way, techniques are still required in which the concentration of compounds of biological interest in small volumes can be measured automatically, with a high degree of precision and in a massive way. Currently, for the quantitative analysis of different substances their concentration is determined by means of optical, electrochemical, radiometric, immunochemical methods, etc. In several cases, prior separation of the analyte of interest is essential, for which the use of chromatographic separation methods is the first option. Such systems are generally coupled to various types of detectors and the optics are the most commonly used. The accuracy and reproducibility of chromatographic techniques have made it the tool of choice both in the industry and in the area of scientific research. However, to gain access to this type of methodologies requires great training and resources, since the degree of sophistication and control of these analysis systems can greatly increase costs. In addition, in many cases the separation and quantification of the samples can be a very time-consuming process (up to half an hour per sample), and this becomes a significant inconvenience if you want to identify a particular substance in a large number of samples, because raises the cost in the separation and analysis process. Derived from the above drawbacks, automatic sampling systems have emerged that facilitate the analysis of substances to some extent.

An alternative for the quantitative analysis of various substances is based on the use of enzymatic or immunochemical reactors, in which thanks to the great biological specificity of these systems, they can react specifically with a particular analyte, producing a derivative with activity electro-active or optical, which can be measured quantitatively by amperometric detection, optical density, luminescence and / or fluorescence, the transduction of this signal being proportional to the concentration of the compound. An example of this is the measurement of glucose concentration through the use of glucose oxidase, glucose is oxidized by the action of glucose oxidase to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide reacts in the presence of peroxidase with 4-hydroxybenzoic acid and with 4-aminoantipyridine to give rise to a red quinonimine dye. The intensity of the color formed is proportional to the glucose concentration and can be measured spectrophotometrically between 460 and 560 nm. The use of chemical concentration gradients can have various uses, for example in chromatography the use of solution gradients with different ionic or organic strength is very common. The isolation and purification of subcellular fractions is another example where various gradients based on sucrose or other solutes are used, in order to produce gradients of different density for the centrifugation of homogeneous solutions of cells and thus be able to separate various subcellular components. Likewise, solutions of concentration of various compounds can be prepared in increasing or decreasing manner, in order to prepare solutions for performing calibration curves.

Discontinuous gradients are formed by carefully placing layers of solutions of different densities in centrifuge tubes. The most widely used method to produce discontinuous gradients is to start with the densest solution and place lower density layers successively. Continuous gradients are prepared from two components with different concentration placed in two reservoirs, connected to each other, fluid from the less concentrated solution is allowed to flow, allowing a similar volume of the concentrated solution to pass, simultaneously control devices are used of the flow rate and mixing for the homogenization of the solution. The frequency of sampling determines the number of points to be obtained in the gradient curve. Automated control in the preparation of gradients has come to solve many of the inconveniences that occur in manual execution, although the level of complexity of the devices, leads to an increase in costs, such is the example of chromatography pumps of high resolution liquids (HPLC) in which the types, flow rates and time of realization of the gradients can be programmed; However, these pumps cost several thousand dollars.

In spite of the numerous advances in the field of gradient production technology, these focus mainly on liquid chromatography, where the volumes to be handled are ml / min, which means a considerable volume if a chromatographic run takes longer or longer. minus 15-20 minutes. With the introduction of various microtechnics in various fields of biology and chemistry, the realization of calibration curves in small volumes is indispensable, especially when all processes are carried out in miniature. Therefore, it is important to design devices that allow such gradients to be performed on scales similar to microtechnics, that is, in the order of microliters or less. Although there are devices to measure accurately around a microliter, they are still manual and frequently handling such volumes leads to a mainly technical error, since it is very difficult to have reproducibility at such small scales.

In order to solve this methodological barrier, a system of generating linear gradients has been designed in a small volume and in a reasonably short time to perform calibration curves with several points, in order to apply these systems to experimentation biochemistry, particularly in the study of enzyme kinetics, where the activity profile is generally studied with no more than 6 increasing concentrations of substrate. In this sense, the The methodological development proposal proposed here will allow to make enzymatic kinetic curves with a large number of points, that is, a greater number of points in a curve, which translates into better study accuracy. The results obtained with this device ensure with a high degree of precision the production of a linear gradient in small volumes.

This proposal is based on the use of two syringes for the construction of said gradients, (one milliliter maximum) mounted on a mechanical system, which is electronically controlled based on stepper motors to produce a programmed differential movement of each piston and thus generate a linear gradient in volumes not larger than one milliliter. With the present prototype, it has been possible to build clearly linear gradients in volumes ranging from 0.010 to 1.00 milliliters, which will allow this methodology to be used in the micrototechnics of analysis recently introduced, both in the field of experimentation and in the industrial field.

SUMMARY OF THE INVENTION

It is an object of the present invention to propose a novel equipment that has the ability to determine the concentration of an analyte in small volumes and in an automated manner. This characteristic is of particular importance for those cases in which the sample quantity is a limitation. Another objective of the system of the present invention whose use makes the amount of reagents used, given the volume required to measure the analyte concentration, is minimal, which provides considerable savings in the analysis and quantification of substances. Another feature of the new system is that it has the feasibility of attaching to existing fluorescence detectors by means of optical fibers, such that the existing digitalization and concentration analysis systems could be used. An additional feature of the invention is that calibration curves can be performed with a linear gradient which provides greater accuracy in the calculation of the problem concentrations.

According to the above, a main objective is to propose a new automatic device for the generation of linear gradients in small volumes that consists of an infusion pump for micro syringes in which the plunger of the micro syringes moves at a set speed and time to make the movement of each piston accelerate or decelerate linearly. The introduction of automatic processes in analytical processes reduces human error and regularly makes the process more efficient.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

Figure 1 illustrates a schematic representation of the capillary fluorescence collection and reading system of the present invention.

Figure 2 is a perspective view of the sample collector carousel.

Figure 3 is a view of the reading system of the invention in which the arrangement of the excitation and reading optical fibers, which are controlled by a CCD fluorescence detector through a computer, is observed.

Figure 4 is an approach view of the system for depositing the sample and the reactor in capillary tubes. Figure 5 is an enlarged view of the capillary tube in which a damping device is observed.

Figure 6 is a screen representation of the reading of the values that result from the implementation of the system for the analysis of a given substance.

Figure 7 is a view showing the new gradient infusion pump specially designed for the present invention.

Figure 8 is a graph showing the calibration curve that reflects the linear relationship of the concentrations tested and the fluorescence measured for the case of a specific example proposed.

Figure 9 is a schematic representation of how the gradient reading was performed in real time in the flow cell for fluorescence measurement.

Figure 10 is representative of the absolute linear relationship that results from plotting the number of speeds with respect to the steps executed per second (Table 1) performed with the new gradient infusion pump.

Figure 11 is a graph that allows us to observe the linear relationship in the measure of the volumes obtained than in a test protocol, according to the calculation estimated by formula 1.

Figure 12 is a view of the mechanism that generates the rotating movement of the sample collection carousel.

DETAILED DESCRIPTION OF THE PREFERRED MODE OF THE INVENTION

This proposal proposes an alternative technological innovation to those already existing, for the identification and quantification of substances through the use of biosensors and enzymatic reactors, which avoids the Chromatographic separation process thanks to the high specificity of enzymes.

One of the modalities of the present invention relates to the automatic generation of gradients in volumes ranging from 0.010 to 1.0 milliliters, with a high degree of precision, which will allow this system to be used in experimentation processes, as well as in the industrial field The analytical procedures use smaller amounts of sample and reagents every time, with the purpose of increasing sensitivity and saving of inputs, from which the need arises to build curves and concentration gradients in the order indicated above. In recent years, the use of microfluidic devices and procedures has been introduced in both the research and industrial area, where the proposed technological development will undoubtedly be of great use.

In a main embodiment of the present invention, it was possible to obtain a linear gradient in a very small volume, which depends on the size of the syringe used (0.010-1.0 ml), so that if you want to make a gradient with the characteristics that here they would be very difficult to achieve this purpose, because in spite of. that in the market there are devices with which a microliter can be measured with precision, it would be impossible to make a gradient in a total volume of 20 microliters with the existing tools, which is equivalent to the automatic mixing of two 10 microliter syringes and especially carried out with absolute temporal precision, that is 580 seconds. Sampling of the resulting gradient can also be done automatically. Given the methodological advances of microtechnics, this procedure will undoubtedly be the tool of choice for performing calibration curves of any standard solution.

In accordance with the embodiment exemplified in Figure 1, the invention consists of a system (100) for automatic collection and reading of samples, in the example described here, on a carousel (55) that has multiple perforations (57) in its periphery, in each of which a sealed capillary tube (60) is placed for the collection of samples of 2.5 μl volume together with a similar volume of enzymatic reactor or chromophore. The capillary tubes (60) can be 1.5mm in length and lmm in internal diameter and function as small cuvettes, in which the fluorescence after the "off-line" collection can be measured, through a measuring system of light (20, 25, 26) or through any other fluorescence detection system, as long as the excitation light can be taken to the corresponding sample and the light emitted through optical fibers is collected (22, 24 ) as illustrated in figure 3.

The carousel (55) is mounted on a base (57) and is rotatably driven by a worm gear drive system (30) (Fig. 12) for which said carousel (55) is coupled to the axis of rotation of the drive system that rotates by means of a cogwheel, which in turn is driven by means of an endless screw. This drive system has a turning ratio of 70: 1, such that for each revolution of the stepper motor (33), to which the collection system is coupled, the carousel rotates 1/70 of revolution. In the present case, the movement control system, both of the carousel and of the sample and reactor deposit systems and of the fluorescence reading system, is controlled by means of an "Allegra" system marketed by Optimal Engineering Systems. Through this control system, the user can program the sampling rate as convenient or use some other automatic movement control system in the market.

The sample deposit system (50) is based on a system of conversion of rotary to linear movement, built on the basis of a linear guide that is coupled to a stepper motor through a belt and pulley geared (Fig. 4). Said linear guide carries with it the deposit system consisting of a stainless steel needle (62), which is attached to a damping device (65) which allows a certain degree of tolerance in the deposit of each sample within the capillary tube (60) without bending the needle (62) deposited or piercing the capillary tube by the pressure exerted. The control and programming of this system is based on a control box of the Allegra brand of the company Optimal Engineering Systems, said system has the necessary instructions and software for programming each of the movements that are required for the automation of this team. For the present case, and without this being interpreted in a limiting sense, it has been programmed for the collection and automatic measurement of up to 120 samples. Collection and reading times can be freely programmed by the user.

The infusion rate of the analyte and reactor is controlled with a pair of automatic infusion pumps (70) that together form a drive system in which each pump motorically pushes the plunger of a respective syringe (72,74) containing the sample to be analyzed and the reactor, to inject them into the capillary tubes (60) of the collection system. Figure 7 shows the structure of only one of the mechanical devices of the drive system of each pump, whose operation is based on the conversion of rotary to linear movement, by coupling a stepper motor (75) with a auger packed screw (76), to which a set (77) is coupled for linear displacement along guides (78), which is the means that exerts the thrust force on the piston of its associated syringe (72,74 ). Both the linear guide assembly (77) and the packed screw (76) of each drive system have the characteristic of producing a movement with zero "play" and with a power transmission increase system, which gives said system of absolute drive precision and repetition in the displacement by rotation or fraction of rotation of the stepper motor.

With regard to the electronic system for the control of movement, in order to obtain a deceleration in the motors that control the device, it was necessary to obtain a formula 1 that allowed to calculate with precision the deceleration speeds with respect to the total time of the process ( translated into the number of steps of the unipolar motor that, for the case exemplified in this application, performs 2175 steps in a span of 9 minutes with 40 seconds) and that is sufficient for the total volume of a calibrated syringe to be displaced. Formula 1 with which the value of the increase in a finite number of set speeds can be obtained is obtained as follows:

X _Í = Í + X _Q = Í + Q = Í

X ₂ = X ₁ - ^ i = i ± i = 2i Xs = X _z + i = i + i - {- i = 3i

X ¹ W ₁ , l = the number of interactions m

I _n = X ₁ + J ₂ + J ₃ + «. +.

H = I m

) ni = í + í2 + í3 + - + mt = Ϊ (1 + 2 + 3 + 4 ». + ni)

Fl = I

Σ rustic —iV) ¹ = ϊ / iW »∑ + i) \} = mimsro totola add I — ι \ ¿j _,, m = in = i Formula 1

where: Xo, is the initial or final speed depending on whether it accelerates or decelerates;

X ₁ is the second speed and so on until Xm is the last speed calculated; ei is the value of each increase. In accordance with the above, and based on a proposed example, the values calculated to achieve a linear acceleration or deceleration in a test protocol of 29 events were those indicated in the following table 1:

TABLE 1

When plotting the number of speeds with respect to the steps executed per second programmed in the designed infusion pump (70), an absolute linear relationship is observed (Fig. 10). Based on the calculated theoretical data, a microcontroller was programmed for each pump (70), so that at the beginning of the gradient generation event, a syringe (72) filled with zero percent solution of the concentration of the Standard will start at maximum speed and the second device with the corresponding syringe (74) containing the standard solution at 100 percent concentration will start the journey at the minimum calculated speed. The speeds were decreasing and increasing, respectively, every 20 seconds simultaneously, so that the total volume of each syringe shifted at the same time (580 seconds). The volume displaced in each syringe was mixed by means of the "Y" connection device (80) and the sample collection was carried out in ependorf tubes at intervals of 20 seconds. The pipe (90) used to carry the analyte and the reactor from each syringe (72.74) to the "Y" connecting device (80) for depositing samples consisted of hollow fused silica tubes, in such a way that the dead volume is not significant for syringes of one milliliter. However, smaller diameter pipe should be used for 0.010 ml syringes.

Infusion pump test and gradient generation. To test the infusion pumps (70) the two syringes (72, 74) were filled separately with vegetable dye and collections were made at the indicated intervals of 20 seconds, so that the volume displaced by each interval was measured, observing and correcting the program until the disappearance of any error. Alternatively, to validate the formation of the gradient, a second protocol was designed, in which, instead of making collections, the mixing was passed inside an online fluorescence reading cell (Fig. 9), in such a way that the gradient formation could be read in real time with a resorufin solution, which fluoresces at 590 nanometers. Readings of the resulting mixture were taken every 5 seconds throughout the event.

Each infusion pump (70) has the ability to dose increasing or decreasing volumes automatically with absolute linearity for the creation of gradients. In figure 11, the linear relationship can be observed in the measure of the volumes obtained which, according to the calculation estimated by formula 1, are absolutely close to the theoretical value, that is, in the test presented in this figure 11 estimated an increase by speed of 2.2 microliters for the syringes calibrated of one milliliter, having to obtain in the last collection 63.8 microliters, having been the linear regression calculated with the values obtained of 0.9978. It is estimated that the small differences are likely due to the measurement of the volumes obtained or differences in the collections, since they were made manually and, as mentioned above, it is very difficult to accurately measure these volumes so small because to the absence of devices in the market for this purpose.

In order to obtain a quantitative reading of the results, an additional test was performed with online reading of a compound that fluoresces at 587 nanometers. The readings in the fluorescent detector (95) were taken in real time, that is, while the gradient was formed, which was introduced to the fluorescence cell, the readings were taken every 5 seconds at a 90 degree angle for excitation and emission in the way it is represented in Figure 9. Some values of the resulting readings are shown graphically in Figure 6. In another alternate embodiment of the invention, a method for collecting samples and reading fluorescence in capillaries is proposed. This method is specially designed to read small volume samples of substances of biological interest, without implying restricting the use of other substances with the ability to emit fluorescence in other areas of both biomedical and industrial interest.

The new method is characterized by the following stages:

1.- Heat sealing of capillary tubes approximately 1.5 mm in diameter x 13 mm in length is carried out, ensuring that they are all approximately the same length.

2. - A series of tubes is placed in a sample carrier device with the desired quantity for the subsequent sample collection; For the case exemplified in the present invention, a carousel carrier with a capacity of 120 samples is described. 3.- The sample deposit system is aligned on the top of the first tube where the first sample will be placed, ensuring that it has the precise height programmed so that the sample deposit is at the bottom of each capillary, without the formation of empty spaces.

4. The sample of interest is flowed through one of the terminals of the Y-device, which may be the substance to be analyzed or a known concentration gradient of a standard solution for the preparation of a calibration curve; The corresponding reactor that will reveal the concentration of the compound of interest or a substance that itself emits fluorescence and that is desired to be quantitatively evaluated is flowed through the other terminal of the Y-device.

5.- Once the previous procedure has been carried out, the program that will place the sample together with the enzymatic reactor and / or fluorophore in each capillary tube is established, that is, the sampling rate and the total number of samples to be collected. 6.- When so required, the reaction time necessary for the development of fluorescence is allowed to pass and then proceed to the reading.

7.- For the reading, the equipment in question has the necessary accessories to carry the excitation light on the top of the first capillary tube, this through an optical fiber (22), the distance from the tube to the fiber can be located according to the characteristics of the sample to be analyzed, with which the size of the optical fiber can be selected, thus selecting the intensity and type of wavelength with which it is desired to excite the sample, The optical instruments can be selected according to the user's convenience, such as the light source and fluorescence detector. The fluorescence emitted by the sample is transmitted to the detector through a second optical fiber (24) placed perpendicular to the capillary tube that will be excited simultaneously, in this equipment an optical detector is used based on a CCD detector of the company "Ocean Optics".

8, - For the reading take, the reading program is selected, which has the same distance of "advance" as for the deposited one, so that, instead of the "collection" the excitation optical fiber will impact the It shows directly on top of each capillary. In this procedure the reading speed can also be programmed, for the described example, 5 seconds are sufficient for each reading, so that the reading of the full carousel is carried out in 6 minutes.

EXAMPLES OF APPLICATION OF THE SYSTEM.

EXAMPLE 1

Substance analyzed: Acetylcholine The measurement of this compound has been carried out with standards of known concentration, the principle consists in the use of the enzymes acetylcholinesterase, choline oxidase and peroxidase. Acetylcholinesterase degrades acetylcholine with choline and acetate production, choline oxidase produces H ₂ O ₂ and betaine. The generated H ₂ O ₂ is identified and quantified by the oxidation of the AMPLEX RED compound in the presence of peroxidase whose derivative is resorufin that has the ability to emit fluorescence at 590 nm when excited with a wavelength of 560 nm; The intensity of the fluorescence is directly proportional to the amount of compound present in each sample.

Taking advantage of the system capacity in automated sample measurement, calibration curves are carried out with the use of the gradient pump. The incubation period with the reactor depends on the analyte to be measured; In this case, the samples deposited in the capillaries are incubated for one hour and subsequently the corresponding fluorescence reading is taken.

EXAMPLE 2 Amplex Red provides a sensitive method to detect hydrogen peroxide as a reaction product of oxidases that generate it. In the test for glutamic acid, it is oxidized by glutamate oxidase to produce α-ketoglutarate, NH3 and hydrogen peroxide. Hydrogen peroxide reacts with Amplex Red in a 1: 1 stoichiometry in the peroxidase catalyzed reaction to generate a highly fluorescent product (resorufin). Resorufin has a maximum absorption and fluorescent emission of approximately 571 nm and 585 nm respectively. With this kit from the house "In vitrogen" can be detected levels as low as 40 nM in a reaction that takes place in 30 minutes.

AMPLEX RED FOR GLUTAMIC Components: Amplex Red

Dimethylsulfoxide (DMSO) Peroxidase (HRP) Hydrogen peroxide (H ₂ O ₂ ) Buffer (Tris-HCl 0.5 M ₅ pH 7.5) Glutamate oxidase (GIuOx) Mpnosodium Glutamic Acid

Solution preparation - Based on the instructions of the Amplex-Red Glutamic Acid / Glutamate Oxidase kit (Invitrogen brand, cat. Al 2221) ₅ in which the preparation of each component is established according to the following relationship: o Amplex Red or Peroxidase: 100 U / ml or 20 mM H ₂ O ₂ : 23 μl of 30% peroxide in 977 μl of dH ₂ O. o Glutamate oxidase (GIuOx): 5 U / ml or 200 mM glutamic acid.

The reactor must contain the following quantities of each component: o HRP: 0.25 U / ml or GIuOx: 0.08 U / ml or Amplex: 100 μM Once the mixture that will form the reactor is made, it is mixed with a similar volume of the sample to be analyzed, which is added simultaneously to each capillary tube, as previously described, that is, 2.5 μl of sample with 2.5 μl of reactor .

Although the present invention has been described on the basis of a carousel-type sample reader, it should be understood that the system is not limited to this particular application but that it can also be used advantageously in a linear displacement reading system or collector.

In the context of the present invention, "sampling" means the process by which the deposit or "injection" of the samples and reactors in the capillary tubes is performed.

Although this invention has been described in the context of the preferred embodiment or embodiment, it will be apparent to those skilled in the art that the scope of the exemplified concept extends beyond the design specifically described and illustrated to other possible alternative modalities of materialization of the invention that are feasible or viable. In addition, although the invention has been described in detail, any expert in the field to which the invention belongs may deduce that some constituent elements of the system may be substituted or others incorporated in the light of the foregoing description without modifying it in essence the result for which it has been conceived.

In view of the above, it will be understood that several elements of the device may be combined with others or replaced by others to conform alternate modes of realization that lead to the same result. Accordingly, it is intended that the scope of the present invention not be construed as limited by the mode particularly described, but rather determined by a reasonable interpretation of the content of the following claims.

BIBLIOGRAPHY.

- Marchini B, De Nuccio L, Mazzei M, Mariottini GL. (2004). A fast centrifuge method for nematocyst isolation from Pelagia noctiluca Forskal (Cnidaria: Scyphozoa). Riv Biol. 97, 505-15.

- Rocklin R. D., Pohl C. A., Schibler J. A. (1987). Gradient elution in ion chromatography. Journal of chromatography. 411, 107-119.

Claims

1. System for collecting and reading fluorescence in capillaries for the identification and quantification of substances in small volumes through the use of biosensors and enzymatic reactors; said system comprises: an automatic sample collection system to be analyzed comprising a mechanism containing multiple perforations, in each of which a sealed capillary tube adapted for the sample collection is placed; a given volume of a sample is collected in each capillary tube together with a similar volume of an enzymatic reactor or chromophore; a sampling device comprising a means for the simultaneous deposition of a reactor and a sample in each capillary tube placed in the sample collection system; said reservoir means consists of a Y-connection device whereby the sample containing the analyte and the enzymatic reactor converge that will develop the reaction for the development and subsequent reading of induced fluorescence; a means of excitation of the sample of interest collected in the capillary tubes of the automatic collection system to apply an excitation signal to induce fluorescence in said sample; a fluorescence detector to measure the fluorescence of the samples that have been subjected to the excitation signal; a drive system for the generation of the gradient, collection and reading of the samples to be analyzed; an electronic control system of the movements necessary for the automatic operation of the different components (gradient generation, deposit, sample change for collection and reading of it); Y a gradient generation system to generate the ratio of calibration curves in small volumes, by calculating the injection rate of the analyte and reactor for mixing them in the Y-connection device of the sampling system.

2. The system of claim 1, wherein the gradient generation system comprises infusion pumps that inject the sample to be analyzed and the reactor into the capillary tubes at predetermined rates.

3. The system of claim 1, wherein the sampling device operates with a rotary to linear movement conversion system, constructed from a linear guide that is coupled to a stepper motor through a belt and pulley in Granada; said linear guide carries with it the reservoir means which is subject to a damping device that allows the deposit of each sample within the capillary tube to be carried out without the reservoir means bending or said capillary tube being punctured by the pressure exerted by said means of deposit.

4. The system of claim 1, which includes an infusion pump drive system consisting of a stepper motor connected with an endless gear which in turn is coupled with a linear sliding assembly with guides; wherein said linear sliding assembly exerts a pushing force on syringes of the sample deposit device and the reactor.

5. The system of claim 1, wherein the collection and reading times are freely programmed by the user.

6. A method for collecting and reading fluorescence in capillaries, which is carried out in a sample collection and reading device in which a plurality of capillary tubes for sample deposition are previously placed; Said method comprises: aligning the sample deposit system on the top of a first capillary tube in which a first sample will be placed, and flowing the sample of interest through one of the branches of a Y-connection device , and a reactor through the other branch of the Y-connection device to reveal the concentration of the sample of interest to deposit them in the capillary tube; calculate the sampling rate and the total number of samples to be collected to program the simultaneous placement of the samples of interest and the reactor and / or fluorophore in the capillary tubes; deposit the rest of the samples and reactors to be analyzed in the capillary tubes; apply to the samples an excitation signal at an intensity and wavelength that is determined according to the type of sample to be analyzed, said excitation signal is applied to the top of each capillary tube; detect the fluorescence emitted by the sample being excited, through a fluorescence detection means placed perpendicular to the sample; and take a reading of the fluorescence value emitted by the samples submitted to the analysis.

7. The method of claim 6, wherein the sample of interest may consist of a substance that itself emits fluorescence.

8. The method of claim 6, wherein the read rate for the reading of the fluorescence emitted by the samples is previously established.

9. The method of claim 6, wherein as many capillary tubes are placed as samples are to be analyzed.

10. The method of claim 6, wherein the precise height of the sample deposit system is calculated to ensure that they are deposited at the bottom of each capillary tube, without the formation of empty spaces.

11. The method of claim 6, wherein the sample of interest may be a known concentration gradient of a standard solution for the preparation of a calibration curve.

12. The method of claim 6, wherein prior to reading, when required, the reaction time necessary for the development of fluorescence is allowed to proceed to then read.

13. The method of claim 6, wherein the sample of interest and the reactor are deposited in the capillary tubes at a speed preselected by gradient pumps.

14. The method of claim 6, wherein the sample of interest and the reactor are deposited in the capillary tubes simultaneously.

15. The method of claim 6, wherein the capillary tubes are heat sealed before being placed in the sampling device for sample collection.

16. The method of claim 6, wherein the positive or negative acceleration rate for gradient formation is calculated by the following formula:

ni —i / = i I = nwnera total to add

«= 1 s = l ^ ^J where: m is the number of speeds or acceleration changes total number to add is the distance traveled by the pistons or the volume displaced in a finite space and time. i is the value of the increment that is added to each speed.