CN1515892A - Micro-analysis chip for absorbance photometric detection and method of use thereof - Google Patents

Abstract

The present invention relates to a microanalysis chip for absorption photometric detection and its application method. It is made by using conventional microanalysis chip channel and coupling it with liquid core waveguide tube. It can implement high-sensitivity absorption photometric detection in microanalysis chip system.

Description

Micro-analysis chip for absorbance photometric detection and method of use thereof

Technical field

The field of the invention relates to microfluidic chip analysis, in particular to a microanalysis chip for absorbing photometric detection and its use method.

Background technique

Microfluidic chip analysis is based on analytical chemistry and analytical biochemistry, relying on micro-electromechanical processing technology, with micro-pipeline network as the structural feature, integrating sample collection, pretreatment, separation, reaction, detection and other parts in several Within an area of square centimeters, the separation, analysis and detection of samples can be completed efficiently and quickly. Since the micro-analysis system was proposed in the early 1990s, Microfluidic Analysis has been at the most active development frontier, representing the development direction of miniaturization and integration of analytical instruments in the 21st century. Usually, a microfluidic chip is composed of upper and lower layers or multi-layer chips, and the material of the chip is single crystal silicon, or quartz, or glass, or high molecular polymer. The area of the chip is about several square centimeters, and the width and depth of the microchannels are on the order of micrometers. Generally, it is a closed microchannel network, and samples enter and exit through holes perpendicular to the chip and connected to the microchannels.

The field that the invention relates to is the research on the detection system on the microfluidic chip. The detection system is an important part of an analysis system. Due to the small size of the chip, the injection volume of the microfluidic chip is only picoliters or nanoliters, and the reaction channel at the detection site is generally tens of microns wide, so the requirements for its detection methods and devices have their own particularities.

Absorption spectroscopy (also known as absorption photometry) is a widely applicable detection method, which occupies an important position in analytical chemistry and is also one of the earliest detection methods used in micro-analysis systems. The wavelength range of photometric detection is about 185-1100 nanometers. However, due to the small detection volume and short absorption path in the channel absorption cell of the microfluidic chip, the detection sensitivity of the absorptiometry is 2-4 orders of magnitude lower than that of the conventional macroscopic system, so the current application in the microfluidic chip analysis system is limited. Very restrictive. In recent years, many studies have been devoted to increasing the absorption path length of the photometric detection system on the microfluidic chip to improve the sensitivity of photometric detection.

The currently published technologies can be divided into the following two categories: one is to increase the optical path of the absorption cell by micromachining, which specifically includes: making a chip with a high aspect ratio channel and directly detecting it perpendicular to the channel. The depth of the channel is generally less than 100 microns , increasing the optical path length by this method has limited effect (Laura Ceriotti, Jan Lichtenberg, et al., Micro Total Analysis Systems 2001, 339-340) making U-shape on chip (Liang Z H, Chiem N, Ocvik G, Tong T, Fluri K, Harrison D J.Anal.Chem.1996, 68:1040) or a flow-through absorption cell with a Z-shaped structure, introducing light and detecting along the channel direction, can obtain a longer optical path, but this structure is difficult to manufacture And alignment, any deviation will cause the loss of analysis signal and the decline of sensitivity, limited by the scattering effect of incident light, the detection optical path should not be greater than 150 microns (HS.Moosavi, Y.Jiang, et al., Electrophoresis, vol.21, pp.1291-1299, 2000); three-layer sandwich chip, the vertical channel absorption cell is made in the middle layer, and its detection optical distance reaches millimeter level, but it is difficult to make the vertical channel in the middle of this chip, and the comparison of sealing the three-layer sandwich chip Difficult and costly (Jeffrey Wolk, MichaelSaid, Morten Jensen, Richard MacReynolds, Knute Stevenson, and Ring-Ling Chien, Micro Total Analysis Systems 2001, 367-368). The other type uses planar waveguide technology to make light total reflection surfaces on the upper and lower sides of the chip. The light is incident from the upper entrance at a certain angle, reflected between the upper and lower reflection surfaces, and can pass through the channel multiple times. The precision requirement is very high, and the incident light needs to be very precise to ensure that the signal is detected in the tiny detection window. The reported detection optical path is 50-272 microns (Moosav HS, Jiang Y, Lester L, McKinnon G, Harrison D J.Electrophoresis , 2000, 21: 1291). There are also integrated specular reflection devices inside the absorption pool, using silicon MEMS etching to make high reflectivity mirrors or reflection channels (Verpoorte E, Manz A, Luedi H, Bruno A E, Maystre F, Krattiger B, Widmer H M, Van der Schoot B H, De Rooij N F. Sens. Actuators. B. 1992, B6:66) (Tiggelaar R M, Veenstra T T, Sanders R G P, Gardeniers J G E, Elwenspoek M C, Van den Berg A. Talanta.2002, 56:331) (Hidekuni Takao, Toshihiko Noda, Mitsuaki Ashiki, Kazuhiro Miyamura, Kazuaki Sawada, Makoto Ishida, Micro Total Analysis Systems 2001, 363-364), based on the multi-plane reflective structure, the reflective optical path is millimeter-scale, Due to the strict requirements on the optical path due to plane reflection, the technical requirements for integrating reflective mirrors in the chip absorption pool are relatively high.

At present, the various methods of increasing the optical length of photometric detection have not broken through the inherent limitations brought about by the tiny size of the chip and its channel, so it cannot really break through the bottleneck of the detection limit, and its detection sensitivity cannot reach the level of conventional spectrophotometers. Moreover, most of them require some highly demanding micro-processing methods, with complex structures and high costs, which limit the application of photometry in the field of chips.

The liquid core waveguide is a kind of optical fiber with liquid as the inner core. In the liquid core waveguide, the light is totally reflected in the waveguide and can be transmitted almost losslessly. This technique has been used in the field of macroscopic photometric detection. Existing studies have shown that using a liquid core waveguide as an absorption cell, its detection absorbance is proportional to the concentration, which conforms to Lambert-Beer's law, and its notable feature is that it can greatly improve the sensitivity of the spectrophotometer and the linear range of measurement. However, in the field of micro-analysis chips, there is no report on the application of liquid-core waveguides in photometric detection.

Contents of Invention

The purpose of the present invention is to break through the inherent limitations brought about by the tiny size of the chip and its channel, provide a micro-analysis chip for absorbing photometric detection and its use method, increase the effective optical path of photometric detection by more than 2-3 orders of magnitude, greatly Increased sensitivity of on-chip spectrophotometric detection.

The micro-analysis chip for absorbing photometric detection provided by the present invention is a micro-analyzing chip for long optical path absorbing photometric detection based on the liquid core waveguide principle. According to the present invention, the absorption pool of the micro-analyzing chip is composed of micro-analyzing chip It is formed by coupling with an external liquid core waveguide.

According to the present invention, the liquid core waveguide is a capillary through which the fluid to be detected flows through the tube, and the properties of the tube wall are used to realize continuous total reflection or reflective transmission of light in the fluid inside the tube. The cross-sectional configuration of the channel in the liquid core waveguide is circular or elliptical. The inner diameter of the inner channel of the liquid core waveguide is in the range of 0.1 micron to 5 mm, and the thickness of the tube wall is in the range of 1 micron to 1 cm. The tube length of the liquid core waveguide is in the range of 5mm-50m.

According to the present invention, in order to realize the waveguide of light in the waveguide, at least one part of the inner wall, outer wall, and tube wall of the waveguide used has the function of reflection or total reflection of light.

According to the present invention, a type of waveguide used is a waveguide for light realized by using a difference in refractive index, and it is characterized in that at least one of the inner wall, outer wall, and tube wall of the waveguide uses a material with a refractive index lower than that of the waveguide. The material of the fluid in the tube is made.

According to the present invention, another type of waveguide used is a waveguide that utilizes the principle of light reflection to realize light. Made of reflective material.

According to another feature of the present invention, the coupling interface between the channel in the micro-analysis chip and the channel of the liquid core waveguide adopts an interface with a small dead volume, which is conducive to reducing the dispersion of the sample strip at the interface, and at the same time, it is not easy to retain air bubbles at the interface to avoid Interfere with normal assay operation.

In the method of using the micro-analysis chip for absorbing photometric detection of the present invention, during actual measurement, the incident light emitted by the light source of the detection system enters the liquid core waveguide from the entrance of the waveguide, passes through the fluid in the tube, is exported from the outlet of the waveguide, and is detected by the photodetector. The light emitted by the light source is absorbed by the fluid in the liquid core waveguide, resulting in a change in absorbance. More advantageously, the incident light of the light source is coaxially aligned with the center of the waveguide, so as to increase the energy of the incident light, that is, improve the signal-to-noise ratio of the measurement. The light exported from the exit of the waveguide is collected by an optical fiber coaxially aligned with the waveguide, and then introduced into a photodetector to improve the efficiency of light collection.

According to the use method of the present invention, the use of a longer waveguide is beneficial to increase the effective optical path of light absorption.

According to the use method of the present invention, relative to the fluid in the waveguide, the outer wall of the tube is used to realize the waveguide, and the effective optical path of photometric detection is lost due to the transmission of light in the tube wall; The outer wall of the tube realizes a waveguide with a longer effective optical path.

According to the use method of the present invention, when the outer wall of the tube is used to realize the waveguide, the tube wall of the liquid core waveguide is thinner, which is beneficial to increase the effective optical path of the absorption pool.

According to the use method of the present invention, for the waveguide adopting the light-transmitting material to make the tube wall, the outer wall of the light entrance of the waveguide is used to cover the light-proof light-shielding material, so as to reduce the entry of useless light that does not pass through the fluid, and can significantly improve the performance of the waveguide. Sensitivity of photometric detection. More advantageously, the outer wall and end surface of the coupling end of the liquid core waveguide and the chip are covered with opaque light-shielding material, which can further improve the sensitivity of photometric detection.

The main advantages of the present invention are: on the small-volume micro-analysis chip, high-sensitivity and high-photometry detection can be carried out; when the required sample volume is in the order of magnitude lower than 1 microliter, the detection sensitivity can reach and exceed the conventional ultraviolet-visible spectroscopic Photometer; in addition, the length of the waveguide is not limited by the size of the chip, and the liquid-core waveguide absorption cell can be as long as centimeters to decimeters, or even meters, to achieve high detection sensitivity; when the liquid-core waveguide is very long , you can use the method of coiling into a spiral to reduce the volume of the system. The light loss of the liquid core waveguide is extremely small, and the anti-interference ability is strong; the detection system has a simple structure, easy processing, stable and reliable performance, small size, and easy integration.

Another outstanding advantage of the present invention is that the absorption pool has relatively simple requirements on the light source, and a small laser or light-emitting diode can be used as the light source, and a strong light detection signal can be obtained without any other light source calibration devices; in order to improve the system Integrative, photodiodes can be used as light detection devices to meet the overall miniaturization requirements.

Another advantage of the present invention is that it can realize continuous injection and detection of multiple samples. The detection sample passes through the liquid core waveguide, that is, it is discharged from the chip system, so it can be continuously injected and used for online monitoring.

The invention can be widely applied to chip-based continuous flow and flow injection analysis.

Description of drawings

Fig. 1 is a micro-analysis chip for long optical path absorption photometric detection based on liquid core waveguide principle according to a preferred embodiment of the present invention.

Figure 2 is a partially enlarged view of the coupling interface between the liquid core waveguide on the chip and the microfluidic chip in Figure 1.

Figure 3. An example of a liquid core waveguide used in a preferred embodiment according to the present invention.

Fig. 4 is a cross-sectional light transmission diagram of the liquid core waveguide used in the embodiment of Fig. 3, comparing the different effects of the light-shielding treatment without light-shielding treatment and the light-shielding treatment.

Fig. 5 is a partial enlarged view of the signal detection end of the liquid core waveguide in the absorption cell according to the preferred embodiment of the present invention.

Fig. 6 shows the record diagram of the detection results of different concentrations of iron (II)-o-phenanthroline complexes by the microchip analysis system equipped with the device of the embodiment of Fig. 3 .

Fig. 7 shows the standard curve of different concentration iron (II)-o-phenanthroline complexes detected by the microchip analysis system equipped with the device of Fig. 3 embodiment and the standard curve diagram of parallel determination in spectrophotometer.

Figure 8. is another example of a liquid core waveguide used in a preferred embodiment according to the present invention.

Fig. 9. Shows the recorded results of the detection of different concentrations of iron (II)-o-phenanthroline complexes by the full Teflon AF liquid core waveguide microchip analysis system equipped with the structure of the embodiment in Fig. 8.

Fig. 10 shows the record diagram of the detection results of the iron (II)-o-phenanthroline complex by the internal silver-plated liquid core waveguide microchip analysis system installed with the structure of the embodiment in Fig. 8.

Detailed ways

Referring to the accompanying drawings, a preferred embodiment 1 according to the present invention will be described in detail below.

Fig. 1 is a structural diagram of a microfluidic chip coupled with a liquid core waveguide manufactured according to a preferred embodiment of the present invention. The microfluidic chip is composed of upper (1) and lower (2) glass sheets, and the thickness of the glass sheets is 1.7 mm. A photomask and a wet etching technique are used to process an "I"-shaped channel on the upper sheet (1), and the microchannel (3) has a width of 100 microns and a depth of 30 microns. Use a 1.7 mm diamond drill bit to drill holes (4), (5) perpendicular to the chip at the end of the "I" shaped channel respectively. The classic high-temperature bonding method is used to realize the permanent sealing of the upper (1) and lower (2) pieces. A microfluidic chip with a length and a width of 30 mm and 20 mm, respectively, was obtained. Close to the position of 2.5mm on the upper transverse channel of the "I" shape, use a diamond glass knife to cut perpendicular to the chip and parallel to the transverse channel to obtain a "T" shaped channel chip. The surface parallel to the transverse channel is ground and polished with fine sandpaper to obtain a transparent plane (8) so that the light source is directly irradiated. A 0.35 mm drill bit is used to drill holes on the chip, and a chip interface (6) with a diameter of about 390 microns is obtained in the outlet channel of the T-shaped channel. The connected waveguide (7) is 5.5 centimeters long. Bond the polytetrafluoroethylene tube with an inner diameter of 0.5 mm to the inlet and outlet (4) and (5) of the microfluidic chip, so as to connect the chip with the external sampling device.

Fig. 2 is a partially enlarged view of the coupling interface between the liquid core waveguide and the microfluidic chip. A Teflon AF1600 liquid core waveguide (7) has an outer diameter of 375 microns and an inner diameter of 50 microns. Use a black paint pen (ZEBRA, Japan) to squeeze out a small drop of paint liquid on the clean glass surface, connect one end of the liquid core waveguide to the pump tube to pump air to prevent the middle channel from entering the paint; the other end is vertically placed in the small paint droplet Give it a dip, pick it up quickly, and pump until the paint is dry. Apply a thin layer of uniform paint (11) on the side wall of the waveguide with a relatively dry paint pen, and the thickness of the paint is about 5-10 microns. The outer diameter of the resulting liquid-core waveguide tip is slightly less than 390 microns. Carefully insert the painted end of the Teflon liquid-core waveguide, and the end of the liquid-core waveguide is tightly bonded to the bottom of the interface. Fix the waveguide with epoxy glue (14). In order to integrate light-emitting diode light sources on the chip, as shown in Figure 2, use diamond drills to penetrate (1) and (2) glass sheets near the interface between the microfluidic chip channel and the waveguide light, grind them with a file, and then polish them with fine sandpaper to obtain A square frame (15) is used to embed the integrated LED light source.

Fig. 3 is a schematic diagram of the optical path of a quartz liquid core waveguide absorption cell coated with a Teflon AF film (9). The refractive index of the Teflon AF film (9) is 1.29, the refractive index of the aqueous solution (10) in the channel is 1.3333, and the refractive index of the quartz wall (8) is 1.51. The total reflection condition of the waveguide used at the entrance of the waveguide is that the incident angle is greater than θ ₀ =75.4°. Light incident along the axis of the channel at an incident angle of less than 15.6° propagates through total reflection in the liquid core waveguide. When there is a light-absorbing substance in the solution, the light is absorbed, resulting in a change in absorbance. It can be seen from Figure 3 that when the incident angle is 15.6°, the optical path through the solution accounts for about one-third of the total optical path. Using pure Teflon AF capillary or total reflection metal capillary on the inner wall can make the optical path greater than or equal to the length of the waveguide; using a waveguide with a very thin quartz wall can reduce the proportion of the optical path of light propagating in the quartz wall, and can also increase the optical path. .

FIG. 4 compares the different effects of the waveguide in FIG. 3 without light-shielding treatment and with light-shielding treatment. A1, A2, B1, and B2 are real microscopic pictures taken by the CCD at the detection end of the Teflon liquid core waveguide. The light source is a 505 nm light emitting diode. A1 and A2 are light distribution diagrams without paint on the incident end surface. B1 and B2 are light distribution diagrams in which the light-shielding treatment shown in FIG. 4 is performed on the incident end. Among them, A1 and B1 were injected with 0.2mM o-phenanthroline-iron(II) chromogenic solution, and A2 and B2 were injected with blank o-phenanthroline solution. It can be seen that when no shading treatment is performed, the solution change does not contribute much to the change of the total light quantity, because most of the light is transmitted through the quartz wall. After shading treatment, the change of the channel center is more obvious. It is proved that the shading treatment helps to reduce the background and improve the detection sensitivity of the system.

Figure 5 shows the design of the liquid core waveguide detection port. A single-core optical fiber (17) with an outer diameter larger than that of the liquid-core waveguide is used at the exit of the liquid-core waveguide, and the light is guided into a photomultiplier tube by the optical fiber for detection. The detection port is also the outlet of the detection solution, and we have designed a diluent pool (16) to keep the environment at the detection outlet stable. Other methods can also be used for detection, such as using a photodiode to directly detect the outlet of the liquid core waveguide, which will greatly simplify the system and achieve overall miniaturization.

Fig. 6 shows the record diagram of the detection results of different concentrations of iron (II)-o-phenanthroline complexes by the microchip analysis system equipped with the device of the embodiment in Fig. 3 . Using the microfluidic chip coupling liquid core waveguide system device in Figure 1 above, and a 505-nm light-emitting diode as a light source, a photomultiplier tube and a low-light measuring instrument as a detector, detect the mixed color of o-phenanthroline-iron (II) solution. The two channels of the T-shaped channel chip are alternately injected with Fe(II) standard series solutions (five solutions in one order of magnitude) and blank solutions.

Fig. 7 shows the standard curve of the detection of different concentrations of iron (II)-o-phenanthroline complexes by the microchip analysis system equipped with the device of the embodiment of Fig. 3 and the standard curve of the parallel determination in the spectrophotometer. The detection range is 2.0-10 micromole/liter standard solution. The curve equation is y=0.0184x+0.0056, R2=0.9991; the curve equation measured in parallel experiments on the spectrophotometer is y=0.011x-0.0028, R2=0.9994. The detected absorbance exceeds the spectrophotometer, but does not reach the minimum value of theoretical calculation. The detection sensitivity is also affected by the monochromaticity of the light source and the incomplete shading of the shading layer.

Fig. 8 is a schematic diagram of an optical path of an absorption cell for liquid core waveguide through internal total reflection. The total reflection tube wall can be a transparent material with a low refractive index or a non-transparent material surface with a high reflectivity.

Fig. 9 is an embodiment adopting the structure of Fig. 8, full TeflonAF liquid core waveguide absorption cell, tube length 6.3 centimeters, to the standard curve that iron (II)-o-phenanthroline complex detects and parallel determination in spectrophotometer Standard Curve Chart. The detection range is 2.0-10 micromole/liter standard solution. The curve equation is y=0.0514x+0.0135, R ² =0.9995, and the curve equation measured in parallel experiments on the spectrophotometer is y=0.0117x-0.0123, R ² =0.9992.

Fig. 10 is another embodiment adopting the structure of Fig. 8, the absorption cell of the inner silver-plated liquid core waveguide, and the record diagram of the detection results of iron (II)-o-phenanthroline complex. Using a 3cm absorption cell, its absorbance can reach 3.7 to 4 times that of a spectrophotometer.

Claims (10)

1. the microanalysis chip that detects of an absorbance is characterized in that, the absorption cell of this chip is coupled by microanalysis chip passage and external liquid core waveguide pipe and constitutes.

2. the microanalysis chip that detects according to the described absorbance of claim 1, it is characterized in that: the liquid core waveguide pipe that adopts in the device, (a) be to remain test fluid in a kind of pipe to be flow through, utilize the character of tube wall to realize light kapillary of transmitting of total reflection or reflection continuously in the pipe internal flow; (b) cross sectional configuration of liquid core waveguide pipe internal channel is circular or oval; (c) internal diameter of liquid core waveguide pipe internal channel is in 0.1 micron-5 millimeters scope; (d) pipe thickness is in 1 micron-1 cm range; (e) pipe range of liquid core waveguide pipe is in 5 millimeters-50 meters scopes; (f) among waveguide inwall, outer wall, tube wall three parts itself, have at least a part to have reflection or total reflection function to light.

3. the microanalysis chip that detects according to claim 1 or 2 described absorbances, it is characterized in that, among the inwall of described waveguide, outer wall, the three of tube wall own, have at least one to adopt refractive index to be lower than the material of tube fluid or to have the material of efficiency light reflecting properties.

4. the microanalysis chip that detects according to claim 1 or 3 described absorbances is characterized in that, applies one deck refractive index at the inwall of described waveguide or outer wall and is lower than the coating of aqueous solution in the pipe or can carries out the coating material that high-level efficiency reflects to light.

5. the microanalysis chip that detects according to claim 1 or 2 described absorbances, it is characterized in that, adopt light transmissive material at described waveguide, light inlet port outer wall at waveguide covers lighttight light screening material, advantageously, coupled end outer wall and the end face at liquid core waveguide pipe and chip covers lighttight light screening material.

6. the microanalysis chip that detects according to the described absorbance of claim 1 is characterized in that, described microanalysis chip passage and the coupling of liquid core waveguide tube passage, and its joint portion sealing, advantageously, the interface of two passages has minimum dead volume.

7. the using method of the microanalysis chip that detects of the described absorbance of claim 1, it is characterized in that, the incident ray of detection system light emitted enters liquid core waveguide pipe by the waveguide inlet, the light that the absorption of fluids light source sends in the liquid core waveguide pipe, thereby the variation of absorbance takes place, derived by the waveguide outlet, photodetector detects.

8. the using method of the microanalysis chip that detects according to the described absorbance of claim 7, it is characterized in that, the incident light of light source and waveguide center of circle coaxial alignment, to improve the incident light energy, light by waveguide outlet derivation, after collecting with the coaxillay aligned optical fiber of waveguide, lead-in light detecting device again is to improve the efficient of collecting light.

9. the using method of the microanalysis chip that detects according to the described absorbance of claim 7 is characterized in that utilizing increases waveguide length, perhaps reduces the pipe thickness of utilizing the pipe outer wall to realize the waveguide of waveguide, improves effective light path of absorption cell.

10. the using method of the microanalysis chip that detects according to the described absorbance of claim 7, it is characterized in that, the dependence pipe outer wall of equal length and internal diameter is realized the waveguide of waveguide relatively, uses the waveguide that relies on inside pipe wall to realize waveguide, can obtain the effective light path of longer photometric detection.

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