TWM480677U - Tandem type photo-detection high-frequency circuit device capable of detecting Cy5 phosphor concentration - Google Patents

Description

Tandem photodetection high-frequency circuit device capable of detecting Cy5 fluorescence concentration

This creation is about a fluorescent detection device, especially when the fluorescent material is excited by the laser light to generate fluorescence, and then the excited fluorescent light is irradiated on the photodetector, which may cause the photocurrent to change or the photodetector to be equivalent. A device that changes the impedance and indirectly affects the frequency response of the high frequency circuit.

The technique related to the present invention is found in the "series photodetection oscillating circuit device" of the national patent No. M329777, which is mainly based on the frequency variation characteristic of a series photodetector frequency circuit (SPFC) of a 48 MHz series. Quantitative analysis of fluorescence concentration was detected. This creation develops a series-type photodetection high-frequency circuit matched by a photodetector, and its output frequency has good analytical sensitivity for the responsivity of the photodetector, and is suitable for fluorescence detection. By matching the high-frequency circuit through different responsive photodetectors, it can be known that in the excitation laser source of the same wavelength, the higher the optical responsivity of the photodetector, the greater the measured frequency change. The lowest fluorescence concentration detection limit of the PT (phototransistor) matched tandem photodetecting device can be measured by the experiment to be 100 amol / 3 μl. According to the different fluorescence concentration of fluorescent dye U1392-HEX 100pmol / 3μl ~ 100amol / 3μl and the frequency of the device, the experimental data can be integrated to establish a linear correlation coefficient R ² = 0.9964. (This paragraph is extracted from the patent bulletin of the creation announcement.)

In recent years, MEMS technology has matured to high molecular polymers. (polymer), bismuth and glass-based biochips are designed using molecular biology, genetic information and chemical analysis, and combined with semiconductor process technology, biochemical technology and precision processing technology to produce high-tech components. A fluorescent detecting device, such as a laser, a filter, a light sensor, etc., is used to form a photodetecting device, and the specificity of the DNA probe is utilized to analyze the heterozygous reaction of the biological sample and simultaneously perform signal processing. The functions of data transmission and data storage have the characteristics of fast analysis, high sensitivity, high specificity and complete experimental data, which can be effectively applied to biomedicine, environmental monitoring and drug development, and improve medical diagnosis ability and effectiveness. Therefore, the world's advanced countries are actively developing biotechnology-related industries, and the huge business opportunities they bring are also limitless. However, there is still a lack of relevant red light detection circuit devices, which is the problem that the creative office wants to solve.

The technical means for solving the problem of the present invention is to propose a new photoelectric detecting device, a red light detecting high frequency circuit, based on the fluorescent detecting principle, and combining the electrical signal measured by the photodetector into the high frequency circuit of the series resonance. In this way, the intensity of the fluorescent signal is converted into a change in frequency, and the frequency variation of different fluorescent concentrations is measured. This device is not only simple, inexpensive, but also has high-frequency and high-sensitivity characteristics. It can detect low fluorescence concentration and establish a linear curve based on different fluorescence and concentration changes. In this creation, a 48MHz red light detection high frequency circuit matched with a photodetector PT is designed to analyze the relationship between the frequency response of the SPFC detection device and the fluorescence concentration, and the frequency detection device is used to measure the fluorescence concentration. The ultimate capability and the basis for the future application of the red light detection high frequency circuit device to the DNA hybrid experiment via the Cy5 firefly cursor.

The equipment for developing the experiments conducted by this creation is as follows.

The structure of the series light detection high frequency circuit device:

Series light detection high frequency circuit device (first figure), including (1) 3mW red laser light source 100 (wavelength 635nm, APCD-635-03, ArimaLASERS, Taiwan) and its illuminated Cy5 fluorescent sample 200 enable the generated excitation of the fluorescent light 300(2) to filter out the filter 400 below 650 wavelength (650FH90-25 , ONSET, Taiwan) (3) self-made 48MHz high frequency oscillating circuit 600, and PT (Phototransistor, TYNTEK, Taiwan) photodetector 500 to match high frequency circuit (4) Agilent 53131A counter 700 (effective frequency range 1Hz ~ 255MHz , Agilent, USA) (5) With the GPIB card computer 800, the program uses the program to read and display the frequency signal on the screen. All detection devices and equipment were placed in a dark room at a temperature of about 25 ° C to facilitate the experimental operation of laser excitation of fluorescence.

Light detection high frequency circuit:

The circuit is based on the Bark-hausen criterion, which is based on (1) must be positive feedback, that is, the input and output phases are different by 0 degree (2) starting frequency, and the open circuit gain is greater than the quartz crystal. The logic gate circuit and the photodetector 500 frame form a series resonant high frequency circuit (second diagram).

Experimental materials and samples:

(1) TE3 buffer (TRIS/pH 8.0 10 mM + EDTA 1 mM, BIO BASIC, USA).

(2) Fluorescent dye: ET996-Cy5 (PURIGO, Taiwan), the optimum absorption wavelength is 635 nm and the optimum fluorescence emission wavelength is 670 nm.

(3) The concentration of Probe ET996-Cy5 diluted to ten times in TE3 buffer is as follows: 100 pmol/5 μl, 10 pmol/5 μl, 1 pmol/5 μl, 100 fmol/5 μl, 10 fmol/5 μl, 1 fmol/5 μl, 100 amol/5 μl, 10 amol/ 5 μl with 1 amol/5 μl.

Serial light detection high frequency circuit device experimental steps:

(1) Place a sample containing a fluorescent dye on a fluorescent detector.

(2) The fluorescent material is irradiated with red laser excitation light of a wavelength of 635 nm to generate fluorescence at a wavelength of 670 nm.

(3) The photodetector 500 is placed above the filter (filtering out excitation light below 650 nm) to detect the excited fluorescent light.

(4) When the excited fluorescent light is irradiated on the photodetector 500, a photocurrent is generated, and the frequency of the high frequency circuit is changed. The frequency data generated by the fluorescent measuring device is extracted and transmitted by the Agilent 53131A counter 700. The GPIB card is connected to the computer 800, and the Agilent VEE 7.5 window program is used to measure the frequency variation of different fluorescence concentrations every five seconds and display the frequency data on the screen.

Photodetector response measurement device:

The photodetector response measuring device comprises: (1) a 175 W xenon lamp source having a color temperature of 5600 °K and a wavelength range of 200 nm to 1100 nm (Model ASB-XE-175-BF, SPECTRAL PRODUCTS, USA). (2) Bandpass filter set (the center wavelength of the filter is 400, 450, 500, 550, 600, 650, and 694 nm, respectively). (3) Standard optical power detection module, including detector (Model 818-UV, NEWPORT, USA) and optical power meter (Model 1815C, NEWPORT, USA).

The photodetector response measurement step is as follows: (1) The light source is matched with the filter to filter out the light sources of each wavelength. (2) The filtered light source measures the luminous flux intensity by a standard optical power detection module. (3) The light detecting element (PT photodetector 500) to be tested is irradiated with each wavelength light source and the photocurrent value thereof is measured, and the optical responsivity of each wavelength is obtained by matching the luminous flux intensity per unit area and the effective illumination area of the element.

Compared with the prior art, the present invention is a novel high-frequency oscillation circuit capable of combining a piezoelectric quartz crystal with a photodetector 500 to detect a change in fluorescence intensity and perform quantitative detection of a Cy5 fluorescence concentration. It can be seen from the experiment that the 48 MHz high frequency circuit device matched by the photodetector PT can measure different fluorescence concentrations.

The tandem photodetection high-frequency circuit device has the characteristics of simplicity, low cost, and high sensitivity, and The higher the basic oscillation frequency, the lower the fluorescence concentration that can be detected by the SPFC device. However, it should be noted that although the SPFC detecting device with a higher basic frequency can detect a lower fluorescence concentration, the frequency of the SPFC device having a higher basic frequency is more disturbed by external noise, that is, the more unstable. The creator will continue to improve the SPFC high-frequency circuit device, expecting to detect lower fluorescence concentrations, and extend to the DNA hybridization experiment to perform qualitative and quantitative analysis of DNA.

1‧‧‧One of the first input gates of the anti-gate

2‧‧‧The first input end of the first anti-gate

3‧‧‧ The first anti-gate output endpoint

4‧‧‧One of the input gates of the second anti-gate

5‧‧‧The second input terminal of the second anti-gate

6‧‧‧The second anti-gate output endpoint

8‧‧‧The output terminal of the fourth anti-gate

9‧‧‧One of the fourth input gates

10‧‧‧The other input terminal of the fourth anti-gate

11‧‧‧The output terminal of the third anti-gate

12‧‧‧One of the third input gates

13‧‧‧The third input terminal of the third anti-gate

100‧‧‧Red laser light source

200‧‧‧Cy5 fluorescent sample

300‧‧‧Inflamed Fluorescence

400‧‧‧ filter

500‧‧‧Photodetector

600‧‧‧48MHz high frequency oscillation circuit

700‧‧‧counter

800‧‧‧ computer

The first figure is a red light detecting high frequency circuit device.

The second picture is a high-frequency circuit diagram for detecting red light.

The third figure is based on (a) PT (b) 3PT (c) KPT (d) DPT and (e) PN and other photodetectors used to match the high-frequency circuit of the sensing element, no sample added and contains fluorescent dye Frequency change plot of the sample (Cy5 100pmol/5μl).

The fourth panel is a plot of the frequency change of the unsampled sample with a sample containing Cy5 100 pmol/5 μl of the fluorescent dye and the reflectance of the five photodetectors.

The fifth figure is a frequency response diagram of a 48 MHz high frequency circuit device matched by a photodetector PT applied to a Cy5 fluorescence concentration of 100 pmol/5 μl to 1 amol/5 μl.

The sixth graph is a linear relationship between the frequency change of the PT light detecting high-frequency circuit device and the Cy5 fluorescence concentration (100 pmol/5 μl to 100 amol/5 μl).

The photodetector 500 and the 48 MHz high frequency oscillating circuit 600 in the first figure are implemented by a series resonant high frequency circuit, as shown in the second figure, wherein the signals are terminated by the first and the first ends. After that, the output is to the terminal 3, and then the series resistor R _{2 is} connected, and then the terminals 4 and 5 are outputted to the terminal 6 through the second back gate, and then the third and the third gates are terminated by the terminals 12 and 13. After that, the output is to the terminal 11, and after the fourth and the gates are terminated by the terminals 9 and 10, the output is to the terminal 8; and wherein the first reverse gate is connected in parallel with the resistor R ₁ and the capacitor C ₁ . the second NAND-based parallel with the resistor R _3, quartz crystal, a photodetector 500 connected in series with the capacitor C ₂ and the inputs and outputs are respectively connected in series to the terminal 1, 2, 6 and endpoint.

The details are as follows.

The equivalent circuit of quartz crystal and photodetector 500:

Photodetector 500 can convert an optical signal into an electrical signal, such as a voltage or current. The photodiode operates in the reverse bias mode, and the photodetector 500 material absorbs photons and emits electrons to generate photocurrent, so that the proportional relationship between the photocurrent magnitude and the input light intensity can be obtained. The photodetector 500 has different types of photodiodes such as PN, PIN, APD, and photoelectric crystal, and the circuit structure is used to explain an equivalent circuit which can be equivalently connected in parallel with the resistor R _p and the capacitor C _p .

The quartz crystal can use an external voltage to generate electric fields on both sides of the wafer. Due to the mechanical and electrical coupling of the piezoelectric material itself, the crystal itself is mechanically deformed, and the cutting surface of the crystal is subjected to mechanical stress. The surface will again produce a potential difference. When an alternating voltage is applied to the upper and lower sides of the piezoelectric crystal, the crystal itself can oscillate at different frequencies. The thickness and direction of quartz crystal cutting determine the resonant frequency ω _r and temperature coefficient caused by its piezoelectric effect. In general, the thinner the resonance frequency is.

Light detection high frequency circuit theory:

So if the drive circuit design using a phase difference θ of the amplifier, the feedback circuit is required having - a phase difference θ to meet the previously mentioned raw Barkhausen oscillation condition. In this creation, the SPFC device uses an amplifier to generate positive feedback oscillations, so the real and imaginary parts of its circuit impedance must meet the phase difference requirement: - θ =tan ^-1 (X/R) (1)

From the equation of equation (1), let A=tan θ be generalized to simplify the following formula:

The correlation between the frequency and the conductance G _p of the photodetector 500 is calculated from the equation (2), and the relationship between the conductance G _p and the angular frequency ω of the photodetector 500 is simplified by Taylor series expansion as follows: Where ω ₀ = 2 πF ₀ ,

F ₀ is the resonant frequency of quartz crystal

Equation (3) is a mathematical function of the frequency response of the SPFC.

The frequency response sensitivity is the differential of the conductance G _p and the angular frequency ω . The creator can calculate the partial differential:

E ₃ =( ω ₀ C _p - AG _p )(2 G _p - ω ₀ C ₀ A )

Further, the optical detector 500 effects the capacitance C _p to the angular frequency ω of the differential, partial differential can also obtain:

From equations (4) and (5), it can be seen that the sensitivity of the SPFC varies with the parameters of the circuit and the equivalent conductance Gp and the equivalent capacitance Cp of the photodetector 500, and versus Both are constants, and the results of (4) and (5) represent that the frequency response of the series-type photodetecting high-frequency circuit device exhibits a linear relationship with the impedance change (Gp and Cp) of the photodetector 500. The experiment of SPFC with high frequency quartz crystal has better sensitivity. Further, if the parameters of the oscillation circuit are fixed, the frequency of the SPFC is simply affected by the conductance G _{p of the} photodetector 500 and the equivalent capacitance C _p .

The implementation results and discussion are as follows.

DNA hybridization is an effective method for comparing genetic differences between different species. It uses a unique pairing pattern between bases on DNA, A pairing with T or pairing with C and G to detect and quantify DNA concentration. In the present creation, the creator proposes a quartz high-frequency oscillating circuit using the photodetector 500 as a detecting element to be coupled to the fluorescent detecting device for quantitatively detecting the fluorescence concentration. Quantitative analysis of DNA concentration can be achieved by using DNA detection experiments with a fluorescent cursor to determine the future detection characteristics of different fluorescence concentrations and high frequency circuit frequency variations.

The photodetector 500, such as a photoconductor and a photodiode, is converted to generate free electron hole pairs by absorbing photons, that is, generating electrons in the conduction band and generating electricity in the valence band. The hole forms a so-called photocurrent effect. Photoelectric crystal Another photodetector 500, which is a structure of a bipolar transistor BJT, has the effect of photocurrent gain, and thus the photorefractive of the photonic crystal is large. The photoelectric crystal is a combination of a photodiode with a reverse bias and a transistor. Compared with the photodiode, the reaction speed is slow but the sensitivity is higher than that of the photodiode. Photovoltaic crystals, like photodiodes, have electron flow and hole currents that affect the impedance characteristics of the optoelectronic crystal through the depletion region. Stated another way, receiving light sources of different intensities by photodetector 500 can also cause impedance variations in the equivalent resistance and capacitance of photodetector 500. In the present creation, the creator mainly performs an experimental analysis of the fluorescence concentration for the SPFC detecting device matched by the photodetector 500.

The photodetector 500 needs to be biased and irradiated with the light source at the receiving end of the photodetector 500 to measure the effect of the photovoltage or the photocurrent, so that the impedance detection can not be directly measured by the impedance analyzer. The equivalent impedance of the device 500. Previously, the creator used the optical responsivity of the photodiode and the optoelectronic crystal to infer the frequency response produced by different photodetectors 500 under different intensity excitations. And it is obtained that under the excitation of the same concentration of fluorescence, the greater the responsivity of the photodetector 500, the more the frequency of the matched high frequency circuit drops.

In this creation, in order to understand the characteristics of the frequency characteristics and the optical responsivity of the series-type light detecting high-frequency circuit, the creator uses the photodetector 500 as a basic sensing element to fabricate a SPFC high-frequency circuit having a frequency of 48 MHz, in different fluorescent colors. Under the conditions of concentration, the following experiments are carried out for explanation and analysis.

48MHz SPFC device applied to red laser module experiment:

HEX fluorescent dye is one of the most commonly used calibration fluorescent dyes for DNA hybridization. Another fluorescent dye most commonly used for DNA hybridization is Cy5 excited by red light source. In this section, the author will attempt to investigate the frequency response of a red laser excitation source applied to Cy5 dye. In order to understand the relationship between the frequency characteristics of the tandem photodetection high-frequency circuit and the optical responsivity, the creator uses five kinds of light. Detector PT (Phototransistor TK124PT, responsivity 135A/W), 3PT (Phototransistor TK168PT, responsivity 92A/W), KPT (Phototransistor TK136PT, responsivity 40A/W), DPT (Phototransistor TK214PT, responsivity 16A/W) and PN (Photodiode TK043PD) ,responsivity 2A/W) is a high-frequency circuit with a frequency of 48MHz for the basic sensing components, and a red laser module as the excitation light source. Under different conditions of fluorescence concentration, the following experiments are explained. analysis:

(1) Basic frequency experiment of Cy5 fluorescence concentration 100pmol/5μl

In order to test whether the fluorescent dye has a fluorescence reaction penetrating filter (650FH90-25, ONSET, Taiwan) after being excited by a red laser, the creator prepared a Cy5 fluorescent dye at a concentration of 100 pmol/5 μl to observe The serial light detects the frequency change of the high frequency device. The same high-frequency circuit matched with five kinds of photodetectors 500 such as PT, 3PT, KPT, DPT and PN is used to observe the frequency difference between the sample without the sample and the sample containing the fluorescent dye. The experimental result is shown in the third figure. The frequency value of the sample containing the fluorescent dye is F1, the frequency value obtained by adding the sample is F2, and the frequency response difference between the fluorescent dye film and the blank film is ΔF=F1-F2, PT The frequency response change ΔF of the SPFC device matched by five photodetectors 500 such as 3PT, KPT, DPT and PN is 135 Hz, 102 Hz, 60 Hz, 46 Hz and 24 Hz, respectively. According to the relationship between the measured frequency change value and the five kinds of photodetector responsiveness (fourth figure), the Cy5 fluorescence concentration of 100pmol/5μl is excited by the red laser, and the generated fluorescence wavelength is indeed The penetration filter changes the photocurrent of the photodetector 500 to affect the frequency. The higher the optical responsivity, the higher the frequency difference obtained by the high-frequency circuit device of the photodetector 500, and the lower the responsiveness, the smaller the frequency difference (ΔF) obtained by the high-frequency circuit device of the photodetector 500.

(2) Fluorescence concentration detection limit of 48MHz series photodetection high frequency circuit

In order to understand the fluorescence concentration limit measured by the energy of the 48MHz high frequency circuit matched by the PT photodetector 500, the creator will mix the Cy5 dye to a different fluorescence concentration of 100pmol/5μl~1amol/5μl in ten-fold dilution. The frequency response of different fluorescent concentrations is measured every five seconds to test the response of the light detecting high frequency circuit. The frequency response obtained from the SPFC device experiment is shown in the fifth figure. Then the frequency value corresponding to the unsampled sample, ~ The frequency values corresponding to the diluted fluorescence concentration of 1 amol/5 μl to 100 pmol/5 μl, respectively. From the fifth graph, for the fluorescence concentration from high to low, the frequency response curve shows a decreasing curve, indicating that the high frequency circuit will have a decreasing frequency change with the decrease of the fluorescence concentration, and the falling frequency response curve and The theoretically derived formula is consistent. It can be seen from the experimental results that the detection limit of the fluorescence concentration of the 48 MHz SPFC with the photodetector PT is 100 amol/5 μl. According to the frequency difference value (ΔF) and the fluorescence concentration (take Log value) of different fluorescence concentration frequencies and unsampled frequencies, a linear regression analysis can be obtained as shown in the sixth figure.

y=0.0557x-17.707 (6)

R ² =0.9958

The embodiment is only one embodiment of the present invention, that is, the 48MHz high-frequency circuit device matched by the photodetector PT can measure different fluorescence concentrations, but the creation can still be made at any time according to the needs. The changes, extensions and applications of the aspects have further expanded their functions, and these are still within the scope of the patent application of this creation.

100‧‧‧Red laser light source

200‧‧‧Cy5 fluorescent sample

300‧‧‧Inflamed Fluorescence

400‧‧‧ filter

500‧‧‧Photodetector

600‧‧‧48MHz high frequency oscillation circuit

700‧‧‧counter

800‧‧‧ computer

Claims (2)

A tandem light detecting high-frequency circuit device capable of detecting a concentration of Cy5 fluorescence, comprising: a light source capable of emitting 3 mW red laser light having a wavelength of 635 nm; a filter positioned above the light source side to filter out light having a wavelength of 650 nm or less, a photodetector disposed above the filter, a 48 MHz high frequency oscillating circuit connected to the photodetector, a 1 Hz to 255 MHz counter connected to the output of the 48 MHz high frequency oscillating circuit, and an interface connected to the counter Cards and busbar computers, in which the photodetector is used in conjunction with high frequency circuits. The tandem photodetecting high-frequency circuit device capable of detecting the concentration of Cy5 fluorescence according to claim 1, wherein the 48 MHz high-frequency oscillating circuit and the photodetector are connected by a series resonant high-frequency circuit, that is, the connected circuit In addition to the photodetector, it also includes four anti-gates, three resistors, two capacitors and a quartz crystal; the first input of the opposite and the gate is connected and the output point is connected in series to the resistor R ₂ , the resistor R _{2 is} also connected in series to the second input of the second anti-gate, the output point is connected in series to the third input of the third anti-gate, and the output point is connected in series to the second input of the fourth anti-gate; The first reverse gate is connected in parallel with the resistor R ₁ and the capacitor C ₁ , the second reverse gate is connected in parallel with the resistor R ₃ , and the quartz crystal, the photodetector and the capacitor C _{2 are} connected in series and the input point after the series connection And the output point is respectively connected to the first input gate of the opposite and the gate and the output of the fourth reverse gate, and the output point is connected to the 1Hz~255MHz frequency counter.