CN106053392B - Device based on micro-nanofluidic reflection interference spectral imaging system and implementation method - Google Patents
Device based on micro-nanofluidic reflection interference spectral imaging system and implementation method Download PDFInfo
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- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
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Abstract
The invention discloses a device based on a micro-nano fluidic reflection interference spectrum imaging system and an implementation method thereof. Compared with the method of adopting a detector as a probe in the prior art, the method of the invention uses the objective lens of the microscope as the optical probe, does not need to be provided with a fixing device for fixing the probe, and improves the light path integration degree of the system; the specific position of the sample is determined through the imaging positioning system, and the detection precision is improved. The invention realizes the real-time switching of the appearance characteristic imaging and the reflection interference spectrum acquisition of the sample and realizes the in-situ dynamic real-time detection of the analysis sample.
Description
Technical Field
The invention relates to a device based on a micro-nanofluidic reflection interference spectral imaging system and an implementation method.
Background
The detection method based on the micro-nanofluidic reflection interference spectral imaging system is convenient and fast, high in precision, can detect a sample in a non-contact and non-destructive manner, and can directly observe and acquire the surface morphology and the reflection interference signals of a trace sample. The reflection interference spectrum imaging technology has practical significance and practical value. For example, in the field of microbiology, reflectance interference spectroscopy imaging systems study the species and quantity of microorganisms by detecting them; in the field of food safety, food safety can be evaluated by monitoring index microorganisms; in the field of ecological monitoring, the method is used for monitoring the change of related microbial flora in ecological environment. In the field of materials, the surface appearance and spectral data such as reflection, absorption, transmission and the like of a sample can be directly observed and collected, and the requirements of most biological and physical experiments can be met. Therefore, the optical sensing detection device based on the reflection interference spectrum imaging system is widely applied in various fields, has considerable market application prospect, and has become a popular research subject at home and abroad.
at present, there are few reports related to devices and implementation methods of the reflection interference spectral imaging system at home and abroad. Wujian Ming project group of Zhejiang university adopts the reflection interference spectrum technology of a porous silicon material micro-fluidic system to detect the growth and metabolism of microorganism escherichia coli (E.coil) in real time, and has the defects that the porous silicon material is unstable in water environment and corresponding PH physiological environment, an optical microscopic imaging system is not available, and the position of the test cannot be accurately positioned. In australia, Dusan et al, porous alumina is used as a substrate material, and a reflection interference spectrum method is used for detecting circulating tumor cells. The method uses the optical fiber probe to detect the sample to obtain the interference spectrum signal, and can not accurately position the test position and the appearance characteristic of the tiny sample.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a device based on a micro-nanofluidic reflection interference spectrum imaging system and an implementation method thereof, which can dynamically observe and collect the appearance and the reflection interference spectrum of a sample on the bottom surface in a micro channel composed of a nanopore array structure in situ and in real time.
In order to realize the task, the invention adopts the following technical scheme:
the device based on the micro-nano fluid control reflection interference spectrum imaging system comprises a micro-flow pump, a micro-fluidic sample cell system, an optical microscope, a C-port light path switcher, a CCD sensor, a computer, a spectrometer and a positioning light source, wherein the C-port light path switcher is connected with the optical microscope, an objective lens of the optical microscope is right opposite to the micro-fluidic sample cell system, the C-port light path switcher is respectively connected with the positioning light source, the CCD sensor and the spectrometer through optical fibers, and the CCD sensor and the spectrometer are both connected with the computer.
further, the liquid flow rate of the micro-flow pump is more than 0.001 ml/min.
Further, the microfluidic sample cell system is of a micro-nano composite structure, and the width and height ranges of the micro-channel are as follows: 10-100 μm.
Furthermore, the microfluidic sample cell system is of a micro-nano composite structure, the bottom of the micro channel is formed by a nano-pore array structure, the diameter of a single nano-pore is 20-180nm, and the thickness of the nano-pore array is 2-10 μm.
Furthermore, the magnification of the objective lens of the optical microscope is 5-100 times, and the numerical aperture is larger than 0.3.
Furthermore, the detection light source is a light source with uniformly distributed light intensity, the power is 1-100W, and the wave band is 200-2800 nm.
Furthermore, the applicable wave band of the C port light path switcher is 200nm-2500nm, and the applicable microscope interface is the C port.
Furthermore, the CCD sensor adopts a high-resolution CCD camera with a zoom lens, the focal length range of the zoom lens is 12-36mm, the pixel number is more than 1024 multiplied by 1024, and the pixel size is less than 5.2 mu m multiplied by 5.2 mu m.
furthermore, the wavelength band of the spectrometer is 200-2800nm, and the signal-to-noise ratio is greater than or equal to 1000: 1.
The method for realizing the reflection interference spectrum imaging based on the device of the micro-nanofluidic reflection interference spectrum imaging system specifically comprises the following steps:
The method comprises the following steps that firstly, a microfluidic sample cell system is fixed on an objective table of an optical microscope, and a microfluidic pump quantitatively injects a sample into the microfluidic sample cell system;
Irradiating light rays emitted by the detection light source onto a sample in the microfluidic sample cell system through an objective lens of the optical microscope, collecting interference light reflected by the sample onto the objective lens, and sequentially passing through the optical microscope and the C-port light path switcher;
Step three, dividing the reflected interference light passing through the C port light path switcher into two beams of light, collecting unit array images of one beam of light through a CCD sensor, transmitting the collected information to a computer through optical fibers, converting the other beam of light through a spectrometer to obtain intensity-wavelength spectrum signals, transmitting the intensity-wavelength spectrum signals to the computer through the optical fibers, and analyzing through a software system to obtain a reflection interference spectrogram;
irradiating light rays emitted by the positioning light source onto a sample through the C-port light path switcher and an objective lens of the optical microscope, converging the sample reflection interference light on the objective lens, collecting unit array images of the converged sample reflection interference light through the C-port light path switcher and the CCD sensor, transmitting the collected information to a computer through optical fibers, and displaying and imaging through the computer; and realizing the reflection interference detection and the appearance imaging of the sample.
Compared with the prior art, the invention has the following technical effects:
1. the invention is provided with the micro-flow pump, which can carry out quantitative control on micro-sampling, thereby realizing dynamic detection.
2. Compared with the means of adopting the probe in the prior art, the objective lens of the microscope is used as the optical probe, a fixing device for fixing the probe is not needed, the possibility of connecting the microscope with an imaging positioning system is provided, and the integration degree of the optical path of the system is improved.
3. In the invention, the light path of the optical interference spectrum system and the light path of the imaging positioning system share one microscope light path, so that the real-time and in-situ detection of the spectrum acquisition and imaging of the sample is realized, and the added imaging positioning system can clearly observe the morphological characteristics of the measured sample.
4. The imaging positioning system is added in the invention, so that the specific position of the sample can be determined, and the detection result is more accurate; and the real-time switching and the real-time detection of the appearance characteristic imaging and the reflection interference spectrum acquisition of the sample are realized.
5. the C-port light path switcher is connected with the positioning light source, the CCD sensor and the spectrometer through optical fibers, and synchronous spectrum acquisition and imaging positioning can be achieved.
In conclusion, the invention realizes the real-time switching and in-situ and real-time dynamic detection of the appearance characteristic imaging and the reflection interference spectrum acquisition of the sample, and the obtained weak signal optical information is comprehensive.
drawings
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a scanning electron microscope topography of a bottom porous nano alumina substrate in a microchannel in a microfluidic sample cell system, wherein (a) is a surface topography; (b) is a sectional view;
FIG. 3 is a reflection interference spectrum of the microfluidic sample cell system detecting single-stranded DNA molecules immobilized on the nanoporous wall, (a) the reflection interference spectrum, (b) the fast Fourier transform spectrum;
FIG. 4 is a reflection interference spectrum of a micro-fluidic sample pool system detection probe single-stranded DNA molecule hybridized with a complementary DNA sequence with single-base mismatch and three-base mismatch, (a) a reflection interference spectrum, and (b) a fast Fourier transform spectrum.
The reference numbers in the figures represent: the system comprises a micro-fluidic pump 1, a micro-fluidic sample cell system 2, an objective lens 3, an optical microscope 4, a C-port light path switcher 6, a CCD sensor 7, a computer 8, a spectrometer 9 and a positioning light source.
The solution according to the invention is explained and illustrated in more detail below with reference to the figures and examples.
Detailed Description
According to the technical scheme, referring to fig. 1, the device based on the micro-nano fluidic control reflection interference spectrum imaging system comprises a micro-flow pump 1, a micro-fluidic sample cell system 2, an optical microscope 4, a C-port light path switcher 5, a CCD sensor 6, a computer 7, a spectrometer 8 and a positioning light source 9, wherein the C-port light path switcher 5 is connected with the optical microscope 4, an objective lens 3 of the optical microscope 4 is over against the micro-fluidic sample cell system 2, the C-port light path switcher 5 is respectively connected with the positioning light source 9, the CCD sensor 6 and the spectrometer 8 through optical fibers, and the CCD sensor 6 and the spectrometer 8 are both connected with the computer 7. The detection data is uniformly collected and analyzed by computer software
The working principle of the device is as follows: light emitted by a detection light source arranged in the microscope mirror reflection device is irradiated on a sample in the microfluidic sample cell system 2 through an optical fiber through an objective lens 3 of an optical microscope 4; the sample reflected interference light is converged on the objective lens 3, and the converged sample reflected interference light sequentially passes through an optical microscope 4 and a C-port light path switcher 5; the reflected interference light passing through the C port light path switcher 5 is divided into two beams of light, one beam of light is subjected to unit array image acquisition through the CCD sensor 6, and the acquired information is transmitted to a computer through an optical fiber and is imaged by corresponding imaging software of the computer; the other beam of light is converted by a spectrometer 8 to obtain an intensity-wavelength spectrum signal, and the intensity-wavelength spectrum signal is transmitted to a computer 7 through an optical fiber and is subjected to data acquisition and analysis by a corresponding spectrum software system of the computer. The reference light path is used for imaging positioning, light emitted by a positioning light source 9 is transmitted to a C-port light path switcher 5 through an optical fiber and is irradiated on a sample through an objective lens 3 of an optical microscope 4; the sample reflected interference light is converged on the objective lens 3, the converged sample reflected interference light passes through the objective lens 3 and the C-port light path switcher 5 of the optical microscope 4, the CCD sensor 6 is used for collecting unit array images, the collected information is transmitted to the computer 7 through optical fibers, and the sample imaging is carried out through corresponding imaging software of the computer.
The device is provided with the micro-flow pump 1, so that quantitative control can be performed on micro-sampling, and dynamic detection is realized; compared with the means of adopting the probe in the prior art, the device of the invention does not need to be provided with a fixing device for fixing the probe, provides possibility for the connection of the microscope with an imaging positioning system and ensures that the integration degree of a light path is higher; in the invention, the light paths of the optical interference spectrum system and the imaging positioning system share one microscope light path, so that the in-situ dynamic real-time detection of the spectrum acquisition and imaging of a sample is realized; the added imaging positioning system can clearly observe the morphological characteristics of the measured sample, and realize real-time switching and in-situ and dynamic real-time detection of the morphological characteristic imaging and the reflection interference spectrum acquisition of the sample.
The micro-flow pump 1 has the liquid flow rate of 0.004 ml/min; the fittings used were plastic nano-tightened fittings and the tubes used were PEEK tubing 1/16 OD.
the microfluidic sample cell system 2 is of a micro-nano composite structure, and the width and height ranges of the micro-channel are as follows: 10-100 μm. The bottom of the micro channel is composed of a nanopore array structure, the diameter of a single nanopore is 20-180nm, and the thickness of the nanopore array is 2-10 μm.
The microstructure is less than 25 μm × 10 μm × 15 μm, the diameter of the nanostructure is less than or equal to 50nm, and the thickness is more than 8 um.
The magnification of the objective lens 3 of the optical microscope 4 is 10 times, and the numerical aperture is 0.7;
The detection light source is a halogen lamp with a uniform light source, the power is 100W, and the wave band is as follows: 350-1100 nm.
in order to ensure that the spectrum collection and the imaging positioning can be carried out synchronously, the applicable waveband of the C-port light path switcher 5 (50% -50%) is 200nm-2500nm, and the applicable microscope interface is a C port.
The CCD sensor 6 adopts a high-resolution CCD camera with a zoom lens, the focal length range of the zoom lens is 12-36mm, the pixel number is more than 1024 multiplied by 1024, and the pixel size is less than 5.2 mu m multiplied by 5.2 mu m.
The spectrometer 8 is a refrigeration type area array back-illuminated spectrometer, collects an optical signal from a sample, and converts an optical interference signal into an intensity-wavelength spectrum signal. The wave band of the spectrometer is 325-1100 nm, and the signal-to-noise ratio is 1000: 1.
In order to determine the specific position of the test sample and enable the detection result to be more accurate, the positioning light source 9 is an HL2000 halogen light source, the wave band range is 350-2500 nm, and the color temperature is 2915K.
The invention discloses a method for realizing reflection interference spectrum imaging by applying a device based on a microfluidic reflection interference spectrum imaging system, which specifically comprises the following steps:
Firstly, a microfluidic sample cell system 2 is fixed on an objective table of an optical microscope 4, and a sample is quantitatively injected into the microfluidic sample cell system 2 by a microfluidic pump 1.
And step two, irradiating the light emitted by a detection light source arranged in a reflecting device of the optical microscope to the sample in the microfluidic sample cell system 2 through an objective lens 3 of the optical microscope 4, and collecting the sample reflected interference light to the objective lens 3 and sequentially passing through the optical microscope 4 and the C-port light path switcher 5.
And thirdly, the reflected interference light passing through the C port light path switcher 5 is divided into two beams of light, one beam of light is subjected to unit array image acquisition through the CCD sensor 6, the acquired information is transmitted to a computer through an optical fiber and is imaged by a corresponding software system, the other beam of light is converted through the spectrometer 8 to obtain an intensity-wavelength spectrum signal, and the intensity-wavelength spectrum signal is transmitted to the computer 7 through the optical fiber and is subjected to data acquisition and analysis by the corresponding software system of the computer.
Step four, light emitted by a positioning light source 9 irradiates a sample through a C-port light path switcher 5 and an objective lens 3 of an optical microscope 4, sample reflection interference light is converged on the objective lens 3, the converged sample reflection interference light passes through the C-port light path switcher 5 and is subjected to unit array image acquisition through a CCD sensor 6, and acquired information is transmitted to a computer 7 through an optical fiber and is displayed and imaged through the computer 7; so far, the reflection interference and the imaging of the sample are realized.
The implementation method of the reflection interference spectrum imaging can accurately position the specific position of the sample and improve the detection precision; and real-time switching, in-situ and dynamic real-time detection of the appearance characteristic imaging and the reflection interference spectrum data acquisition of the sample are realized.
example (b):
embodiments of a device based on a micro-nanofluidic reflection interference spectral imaging system and an implementation method are given below.
in this embodiment, the device based on the micro-nanofluidic reflection interference spectral imaging system includes a microfluidic pump 1, a microfluidic sample cell system 2, an optical microscope 4, a C-port optical path switcher 5, a CCD sensor 6, a computer 7, a spectrometer 8, a positioning light source 9, and a detection light source.
A microflow pump with the flow rate range of 0.004 ml/min; the microfluidic sample cell system 2 is of a micro-nano structure, the micro-structure is 25 mu mx10 mu mx15 mu m, the diameter of the nano structure is 50nm, and the thickness of the nano structure is 8 mu m; the magnification of the objective lens 3 of the optical microscope 4 is 10 times, and the numerical aperture is 0.70; the detection light source of the optical microscope 4 is a halogen lamp with a uniform light source, the power is 100W, and the wave band: 350-1100 nm; the applicable wave band of the C port light path switcher 5 (50% -50%) is 200nm-2500 nm; the applicable microscope interface is a port C; the CCD sensor 6 adopts a high-resolution CCD camera with a zoom lens, the focal length range of the zoom lens is 12-36mm, the pixel number is 1024 multiplied by 1024, and the pixel size is 5.2 mu m multiplied by 5.2 mu m; the spectrometer 8 is used for testing a sample, and the refrigeration type area array back-illuminated spectrometer collects an optical signal from the sample and converts the optical interference signal into an intensity-wavelength spectrum signal. Wherein the wavelength band of the spectrometer is: 325-1100 nm; the signal-to-noise ratio is 1000: 1; the positioning light source 9 is used for detecting the specific position of the optical detection sample, the light source is an HL2000 halogen light source, and the wave band range is as follows: 350-2500 nm; color temperature: 2915K.
The implementation method of the optical interference of the embodiment specifically includes the following steps:
step one, adopting a porous nano anodic aluminum oxide nano structure with the diameter of 50nm and the thickness of 8 μm and PMMA with the thickness of 25 μmx10 μmx15 μm to be combined through an anodic bond to serve as the microfluidic sample cell system 2. The sample single-chain DNA enters the microfluidic sample cell system 2 from a pipette and is fixed in the cell to be used as a probe, and the microfluidic pump 1 quantitatively enters the sample complementary DNA into the microfluidic sample cell system 2 at the speed of 0.004 ml/min;
secondly, a detection light source is a 1000W halogen lamp, emergent light passes through a reflection light path of an optical microscope 4 and passes through an objective lens 3 with the magnification of 10 times and the numerical aperture of 0.70 to irradiate on a nano porous alumina sample with the thickness of about 8 mu m; the sample reflected interference light is converged on an objective lens 3, and the converged light passes through an optical microscope 4 and a C port light path switcher 5 (50% -50%);
Step three, the converged light is divided into two beams of light through a C-port light path switcher 5, and one beam of light is converted through a CCD sensor 6 with the pixel number larger than 1024 multiplied by 1024 and then transmitted to a computer 7 through an optical fiber; the other beam of light is transmitted into the spectrometer 8 through a micro-area optical fiber with the wave band range of 360-2500 nm, the core diameter of 600 mu m and the length of 2.0m, and the information is transmitted to the computer 7 through the optical fiber after the light is converted in the spectrometer 8;
Step four, a halogen lamp positioning light source 9 with the power of 1000W irradiates the sample through a C-port light path switcher 5 and an objective lens 3 of an optical microscope 4; the sample reflected interference light is converged on the objective lens 3, the converged sample reflected interference light passes through the C-port light path switcher 5, is converted by the CCD sensor 6 and then is transmitted to the computer 7, and the image is displayed in the computer 7; therefore, the reflection interference spectrum and the imaging of the sample based on the reflection interference spectrum imaging system are realized.
experimental analysis:
Fig. 2 is a Scanning Electron Microscope (SEM) surface topography and a cross-sectional view of the apparatus of the present invention acquiring the porous nano anodized aluminum substrate of the microfluidic sample cell system of the above-described embodiment.
eff effFig. 3 is a reflection interference spectrogram of a microfluidic sample cell system for detecting single-stranded DNA molecules immobilized on walls of nanoporous wells, a reflection interference spectrogram identified by a spectrum, and an FFT spectrogram processed by Fast Fourier Transform (FFT) data, wherein the abscissa of a peak corresponds to an Effective Optical Thickness (EOT) spectrogram for detecting the solidification condition of the single-stranded DNA of the sample in the microfluidic sample cell system, (a) and (b) in fig. 3, and line segments represent AAO + Au and probe DNA respectively from high to low according to the peak value, (a) in fig. 3 is an in-situ reflection interference spectrum (RIFS) of the substrate and the probe DNA molecules, and the RIFS of the DNA probe molecules moves to the right relative to the substrate material as the refractive indexes n and L change as the DNA molecules solidify on the walls of the substrate wells, and the signal-to-noise ratio and resolution of the RIFS can be more visually observed as the interference molecules enter the walls of the nanoporous wells, see fig. 3 b for the change of the optical molecules caused by the refractive indexes n and L, and the FFT.
FIG. 4 is a reflection interference spectrum of the probe DNA hybridized with a complementary DNA sequence having three-base mismatch and single-base mismatch and a Fast Fourier Transform (FFT) spectrum processed by FFT data, wherein the abscissa of the peak corresponds to an Effective Optical Thickness (EOT) spectrum to detect the interaction between DNA molecules in a microfluidic sample cell system, obtained by the device of the present invention; in the (a) and (b) graphs in fig. 4, the line segments represent the compDNA, MMC and MM3C, respectively, from high to low in terms of peak values. FIG. 4 shows the capture of complementary DNA molecules by probe DNA molecules and complementary DNA single strands with three-base mismatches and one-base mismatches, for use in practical assays. From the (b) graph, we found that different base mismatches can cause different changes, thereby illustrating the feasibility, sensitivity and specificity of the RIFS technology.
Claims (2)
1. the device based on the micro-nano fluidic control reflection interference spectrum imaging system is characterized by comprising a micro-flow pump (1), a micro-fluidic sample cell system (2), an optical microscope (4), a C-port light path switcher (5), a CCD sensor (6), a computer (7), a spectrometer (8) and a positioning light source (9), wherein the C-port light path switcher (5) is connected with the optical microscope (4), an objective lens (3) of the optical microscope (4) is right opposite to the micro-fluidic sample cell system (2), the C-port light path switcher (5) is respectively connected with the positioning light source (9), the CCD sensor (6) and the spectrometer (8) through optical fibers, and the CCD sensor (6) and the spectrometer (8) are both connected with the computer (7);
the magnification of an objective lens (3) of the optical microscope (4) is 5-100 times, and the numerical aperture is larger than 0.3;
The liquid flow rate of the micro-flow pump is more than 0.001 ml/min;
the microfluidic sample cell system (2) is of a micro-nano composite structure, and the width and height ranges of the micro-channel are as follows: 10-100 μm;
the microfluidic sample cell system (2) is of a micro-nano composite structure, the bottom of a micro channel is formed by a nano-pore array structure, the diameter of a single nano-pore is 20-180nm, and the thickness of the nano-pore array is 2-10 mu m;
The applicable wave band of the C port light path switcher (5) is 200nm-2500nm, and the applicable microscope interface is a C port;
the CCD sensor (6) adopts a high-resolution CCD camera with a zoom lens, the focal length range of the zoom lens is 12-36mm, the pixel number is more than 1024 multiplied by 1024, and the pixel size is less than 5.2 mu m multiplied by 5.2 mu m;
the wavelength band of the spectrometer (8) is 200-2800nm, and the signal-to-noise ratio is greater than or equal to 1000: 1.
2. The method for realizing the reflection interference spectrum imaging based on the micro-nanofluidic reflection interference spectrum imaging system in the claim 1 is characterized by comprising the following steps:
Firstly, a microfluidic sample pool system (2) is fixed on an objective table of an optical microscope (4), and a sample is quantitatively injected into the microfluidic sample pool system (2) by a microfluidic pump (1);
secondly, irradiating light rays emitted by a detection light source onto a sample in the microfluidic sample cell system (2) through an objective lens (3) of an optical microscope (4), collecting interference light reflected by the sample onto the objective lens (3), and sequentially passing through the optical microscope (4) and a C-port light path switcher (5);
step three, the reflected interference light passing through the C port light path switcher (5) is divided into two beams of light, one beam of light is subjected to unit array image acquisition through a CCD sensor (6), the acquired information is transmitted to a computer through an optical fiber, the other beam of light is converted through a spectrometer (8) to obtain a spectral signal with intensity and wavelength, the spectral signal with intensity and wavelength is transmitted to a computer (7) through the optical fiber, and a reflected interference spectrogram is obtained through software system analysis;
Irradiating light rays emitted by a positioning light source (9) onto a sample through a C-port light path switcher (5) and an objective lens (3) of an optical microscope (4), collecting reflected interference light of the sample on the objective lens (3), collecting unit array images of the collected reflected interference light of the sample through the C-port light path switcher (5) by a CCD sensor (6), transmitting the collected information to a computer (7) through optical fibers, and displaying and imaging through the computer (7); and realizing the reflection interference detection and the appearance imaging of the sample.
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