A device for rapid proliferation and detection of microorganisms

Abstract

The application discloses a kind of microbial rapid proliferation and detection device.In the device, the mixed solution of microorganism and nutrient solution can be made to flow continuously by microfluidic chip channel control.Circulating process, microorganism in mixed solution is settled on the surface of sensor substrate in chip settling chamber.In addition, through liquid flow, microorganism on the surface of sensor substrate can exchange nutrient substance and metabolite with mixed solution in time, and grow and proliferate rapidly, so that the sensor can detect the presence of microorganism in blood or urine in a short time.The rapid proliferation and detection device combined with microfluidic chip and optical sensor can concentrate and concentrate microorganism in blood or urine in the local area of sensor substrate surface, so only a small amount of sample is needed to realize detection, which greatly reduces the amount of blood and urine sample required for detection.In addition, the optical sensor converts the number of microorganism deposited on the surface of sensor substrate into the signal of spectral peak displacement in real time, so as to realize real-time monitoring of the proliferation process of microorganism.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection instruments, and more particularly to a blood or urine microorganism rapid proliferation and detection device. BACKGROUND

[0002] Blood microorganism infection can cause patients to develop bacteremia, fungemia and septicemia, which seriously endanger the life and health of patients. Due to the diversity of microorganisms, blood disease infection needs to timely determine the type of infection bacteria so that the clinic can give targeted drug treatment to patients; detecting microorganisms in the blood of patients has important clinical significance for the diagnosis, treatment and prognosis of bacteremia, fungemia and septicemia. Since the blood of patients with blood microorganism infection contains a very low amount of bacteria, generally about 10 CFU / mL, it is difficult to detect microorganisms at such a low concentration regardless of which traditional detection method is used. In addition, urine microorganisms can reflect urethral infection and most acute prostatitis. Urinary tract infections caused by microorganisms such as bacteria and fungi often lack obvious symptoms, among which the elderly patients are particularly susceptible. Isolating and culturing pathogenic bacteria is also very important for the diagnosis of urinary tract infection. At present, the culture method is still the gold standard for the diagnosis of blood or urine disease infection, but in actual application, the following problems exist:

[0003] (1) A large amount of sample is required. The blood culture method requires 10-20 mL of venous blood sample from patients. For children and critically ill patients, the extraction of a large amount of venous blood will affect the nutrient delivery and recovery of the patient.

[0004] (2) The detection time is long. A large amount of time is consumed in blood or urine culture to wait for the growth and proliferation of bacteria, and it generally takes 24-48 h to detect positive, and the detection time required for fungi is even longer, which seriously delays the timely diagnosis and treatment time of blood or urinary microorganism infection patients.

[0005] (3) Manual operation is complicated. After culture, bacteria or fungi are generally inoculated on agarose culture medium by manual operation, and then single colonies are selected by manual operation, and mass spectrometry analysis or biochemical detection is performed to determine the type of bacteria or fungi. In this process, complicated manual operation is involved, which increases the error caused by operation and even affects the accuracy of the final detection result.

[0006] Traditional culture method needs to consume a large number of samples, time and complicated manual operation, how to shorten the culture time and identify the microorganism species in time is the most urgent thing in blood-borne disease and urinary tract infection disease. Chinese patents CN107177496A and CN106244438A disclose blood microorganism culture vessels, but there are still problems of long culture time and the need for separation and detection after culture; Chinese patents CN113073096A and CN111500460A disclose methods for separating, purifying and enriching pathogenic microorganisms in blood, although the pathogen enrichment can be directly performed using the blood sample without blood culture, but still needs a large amount of blood sample to achieve, and needs to be detected after enrichment, and cannot realize detection during microorganism proliferation. SUMMARY

[0007] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a microorganism rapid proliferation and detection device.

[0008] The second purpose of the present application is to provide the application of the microorganism rapid proliferation and detection device.

[0009] The above-mentioned purpose of the present application is realized by the following technical scheme:

[0010] A microorganism rapid proliferation and detection device, comprising a light source, a peristaltic pump, a hose, a microfluidic chip, a microorganism sedimentation chamber, an optical sensing substrate and a spectrometer;

[0011] The microfluidic chip comprises a transparent cover plate and a microfluidic channel layer, one end of the microfluidic channel layer is provided with a liquid inlet, and the other end is provided with a liquid outlet; a microfluidic straight channel is arranged between the liquid inlet and the liquid outlet, the middle part of the microfluidic straight channel is a hollow structure, and penetrates through the microfluidic channel layer, and the size thereof is matched with the opening size of the microorganism sedimentation chamber; the hose is connected with the liquid inlet and the liquid outlet of the microfluidic chip through the peristaltic pump;

[0012] The microorganism sedimentation chamber is located below the hollow structure of the microfluidic chip and is in communication with the microfluidic straight channel, the optical sensing substrate is arranged at the bottom of the microorganism sedimentation chamber, and the bottom of the microorganism sedimentation chamber is a transparent structure;

[0013] The light source and the spectrometer are respectively located on the upper and lower sides of the microfluidic chip and the microorganism sedimentation chamber, and the light source, the microfluidic chip, the microorganism sedimentation chamber and the spectrometer are on the same light path.

[0014] The device of the present application is connected to the fluid channel of the microfluidic chip by a hose, and forms a closed solution circulation channel with the microbial sedimentation chamber. The cross-sectional area of the microbial sedimentation chamber is much larger than that of the microfluidic straight channel. When the liquid in the microfluidic straight channel passes through the sedimentation chamber, the flow rate decreases due to the sudden increase in cross-sectional area, and thus the large particles in the fluid, such as bacteria or fungi, will settle under the action of gravity and eventually enrich on the surface of the optical sensor substrate. The blood or urine microorganisms are mixed with the culture solution and continuously circulated in the microfluidic device by a peristaltic pump. After passing through the sedimentation chamber of the device, the microorganisms in the blood or urine are settled and aggregated on the surface of the sensor substrate, thus greatly reducing the required amount of patient blood or urine sample. As the solution continuously flows, the nutrients in the solution continuously contact and exchange with the metabolites of the microorganisms at the bottom, allowing the microorganisms on the surface of the sensor substrate to rapidly grow and proliferate, thereby increasing the concentration and enabling the optical sensor to detect the presence of microorganisms in the blood or urine in a relatively short period of time. By continuously collecting the transmission spectrum of the optical sensing substrate at different time points until the peak shift of the collected spectrum changes significantly, it is determined that microorganisms are present in the original blood or urine. The device uses a high-sensitivity optical sensor substrate and enriches the microorganisms in the blood or urine mixture by sedimentation to increase the local concentration of the sensor, thereby shortening the detection time of microorganisms and monitoring the proliferation process of microorganisms.

[0015] Preferably, the hose is a soft silicone catheter.

[0016] Preferably, the microfluidic chip is a PDMS microfluidic chip, which includes a PDMS cover plate and a PDMS microfluidic channel layer.

[0017] Preferably, the microbial sedimentation chamber includes a chamber layer consistent with the size of the microfluidic chip and a transparent bottom plate. The chamber layer is a substrate with a hollow structure in the middle, and the hollow structure is sealed and connected to the transparent bottom plate to form the microbial sedimentation chamber.

[0018] Preferably, the optical sensing substrate is a noble metal sensing array with localized surface plasmon resonance (LSPR) effect that is sensitive to the surrounding refractive index. The rapid proliferation and detection device combining the microfluidic device with the LSPR sensor can concentrate and concentrate the microorganisms in the blood or urine on the surface of the sensor in a local area, greatly reducing the required amount of patient blood or urine sample. In addition, the LSPR sensor converts the number of microorganisms deposited on the surface of the sensor substrate into a LSPR spectrum peak shift signal in real time, thereby realizing the detection of the proliferation process of microorganisms. The high sensitivity and real-time characteristics of the LSPR sensor further shorten the detection time of microorganisms in blood or urine.

[0019] Further preferably, the noble metal sensing array is a three-dimensional gold nanopillar array, a gold nanohole array or a gold nanomushroom head array.

[0020] Further preferably, the noble metal sensing array is composed of an optically transparent substrate and a gold layer with nanometer thickness.

[0021] Preferably, the optical spectrometer is an ultraviolet-visible spectrometer.

[0022] Preferably, the optical sensing substrate is modified with specific antibodies of the microorganism to be detected, so as to further distinguish the type of microorganism.

[0023] Preferably, the peristaltic pump is a commercial peristaltic pump, which can adjust the liquid peristaltic speed of the device.

[0024] The application also provides the use of any of the above devices in rapid proliferation and detection of microorganisms. The microorganisms include but are not limited to bacteria and fungi, and the samples for detection include but are not limited to blood and urine. For example, the use in rapid proliferation and detection of blood microorganisms.

[0025] A method for rapid proliferation and detection of blood microorganisms, comprising the following steps:

[0026] S1, mixing 200 μL of venous blood of a patient with a microorganism nutrient solution in a volume ratio of 1:5, and injecting the mixture into a silicone hose of the detection device, the silicone hose being connected with a fluid channel and a microorganism sedimentation chamber, and an optical sensing substrate being placed at the bottom of the microorganism sedimentation chamber. After the mixed solution fills the entire channel of the device, the fluid channel of the device is closed, and the detection device is placed in a 37℃ incubator;

[0027] S2, turning on the switch of the peristaltic pump and adjusting the flow rate, so that the mixed solution in the hose enters the sedimentation chamber of the device at a low speed and circulates, and the microorganisms and other large particles in the mixed solution are sedimented in the sedimentation chamber and enriched on the surface of the optical sensor substrate at the bottom of the sedimentation chamber;

[0028] S3, continuously collecting the transmission spectrum of the optical sensing substrate at different time points until the peak position of the collected spectrum shows a significant change, which indicates that the microorganism in the original blood is detected. The device uses a high-sensitivity optical sensor chip and sedimentation and enrichment of microorganisms in the blood mixture to increase the local concentration of bacteria on the sensor, thereby shortening the detection time of microorganisms.

[0029] In step S1, when the microorganism is bacteria, the nutrient solution generally refers to trypsin-digested soybean broth, brain heart infusion broth, Columbia broth, etc., and the required nutrient solution is selected according to the requirements of the bacteria to be detected for nutrition.

[0030] The present application is based on the microfluidic channel for solution microorganism enrichment and the high sensitivity response of LSPR sensor to the refractive index change of the sensing layer, so that the detection device has the advantages of small required sample volume, high sensitivity, short detection time and simple operation.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The present application discloses a kind of microorganism rapid proliferation and detection device.In the present device, by microfluidic chip channel control can make microorganism and nutrient solution mixed solution constantly circulating flow.In the circulation process, microorganism in mixed solution is deposited on the sensor substrate surface in chip sedimentation chamber.In addition, through liquid flow, microorganism on the sensor substrate surface can be in time with mixed solution exchange of nutrient substance and metabolite, and rapidly grow and proliferate, so that sensor can detect the presence of microorganism in blood or urine in a short time.The rapid proliferation and detection device of microfluidic chip and optical sensor combination can concentrate and concentrate microorganism in blood or urine in the local area of sensor substrate surface, so only a small amount of sample can realize detection, which greatly reduces the amount of blood and urine sample required for detection.In addition, optical sensor converts the number of microorganism deposited on the sensor substrate surface into the signal of spectral peak displacement in real time, so as to realize real-time monitoring of microorganism proliferation process. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is the schematic diagram of the whole structure of the present application microorganism rapid proliferation and detection device.

[0034] Figure 2 It is the channel size and fluid velocity simulation diagram of the present application microorganism rapid proliferation and detection device.

[0035] Figure 3 It is the LSPR spectrum diagram and LSPR spectrum peak displacement diagram in the process of Candida albicans proliferation.A: different time spectrum diagram, B: P point peak displacement changes with time diagram.

[0036] Figure legend: 1-light source; 2-peristaltic pump; 3-hose; 4-microfluidic chip; 5-microorganism sedimentation chamber; 6-optical sensing substrate; 7-spectrometer; 41-transparent cover plate; 42-microfluidic channel layer; 51-cavity layer; 52-transparent bottom plate. DETAILED DESCRIPTION

[0037] The present application will be further described below in conjunction with the drawings and specific examples of the present application, but the examples do not limit the present application in any form.The reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field, unless otherwise specified.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] Example 1: A device for rapid proliferation and detection of microorganisms

[0040] like Figure 1 As shown, a device for rapid proliferation and detection of microorganisms includes a light source 1, a peristaltic pump 2, a flexible tube 3, a microfluidic chip 4, a microbial sedimentation chamber 5, an optical sensing substrate 6, and a spectrometer 7.

[0041] The microfluidic chip 4 includes a transparent cover plate 41 and a microfluidic channel layer 42. One end of the microfluidic channel layer 42 is provided with an inlet and the other end with an outlet. A microfluidic straight channel is provided between the inlet and the outlet. The middle of the microfluidic straight channel is hollow and runs through the microfluidic channel layer 42. Its size matches the opening size of the microbial sedimentation chamber 5. The flexible tube 3 is connected end to end to the inlet and outlet of the microfluidic chip 4 through a peristaltic pump 2.

[0042] The microbial sedimentation chamber 5 is located below the hollow structure of the microfluidic chip 4 and is connected to the microfluidic straight channel; the microbial sedimentation chamber 5 includes a chamber layer 51 of the same size as the microfluidic chip 4 and a transparent base plate 52; the chamber layer 51 is a substrate with a hollow structure in the middle, and its bottom is sealed to the transparent base plate 52 to form the microbial sedimentation chamber 5; the optical sensing substrate 6 is placed at the bottom of the microbial sedimentation chamber 5.

[0043] The light source 1 and the spectrometer 7 are located on the upper and lower sides of the microfluidic chip 4 and the microbial sedimentation chamber 5, respectively, and the light source 1, the microfluidic chip 4, the microbial sedimentation chamber 5 and the spectrometer 7 are on the same optical path.

[0044] The device is connected end-to-end to the fluid channel of the microfluidic chip via a flexible tube, forming a closed solution circulation channel with the microbial sedimentation chamber. The cross-sectional area of ​​the microbial sedimentation chamber 5 is much larger than the cross-sectional area of ​​the microfluidic straight channel (e.g., Figure 2 A) When liquid passes through the settling chamber, the flow velocity decreases due to the sudden increase in cross-sectional area (e.g., Figure 2B), so that the large particles in the solution, such as bacteria, etc. will be settled under the action of gravity and finally enriched on the surface of the optical sensor substrate. The blood or urine microorganisms are continuously circulated with the culture mixed solution by the peristaltic pump, and the microorganisms in the blood or urine are settled on the surface of the sensor substrate after passing through the settling chamber of the device, which greatly reduces the required amount of patient blood or urine sample. With the continuous flow of the solution, the nutrients in the solution are continuously in contact with and exchanged with the nutrients of the microorganisms at the bottom, so that the microorganisms on the surface of the sensor substrate proliferate rapidly and the concentration increases, so that the sensor can detect the presence of microorganisms in the blood or urine in a short time. By continuously collecting the transmission spectrum of the optical sensing substrate at different time points until the peak position of the collected spectrum changes significantly, i.e. the presence of microorganisms in the original blood or urine is detected.

[0045] In particular, the hose 3 is a soft silicone catheter.

[0046] In particular, the microfluidic chip 4 is a PDMS microfluidic chip, which includes a PDMS cover plate and a PDMS microfluidic channel layer.

[0047] In particular, the optical sensing substrate 6 is a noble metal sensing array (LSPR sensor substrate) with a surrounding refractive index sensitive local surface plasmon resonance effect. It is composed of an optically transparent substrate and a gold layer with a nanoscale thickness.

[0048] In particular, the spectrometer is an ultraviolet-visible spectrometer.

[0049] Embodiment 2: A method for rapid proliferation and detection of fungi

[0050] Using the device described in Embodiment 1, this embodiment provides a method for rapid proliferation and detection of fungi, which includes the following steps:

[0051] S1. Mix a certain concentration of 200 μL of Candida albicans solution simulating blood infected patient sample with Sabouraud culture solution according to a volume ratio of 1:5, and inject it into the soft silicone catheter of the detection device, wherein the soft silicone catheter is connected with the microfluidic channel, the peristaltic pump and the microbial settling chamber, and the LSPR sensing substrate is placed at the bottom of the settling chamber.

[0052] S2. After the mixed solution fills the entire device channel, place the detection system in a 37°C constant temperature incubation system, turn on the peristaltic device of the detection device, and make the mixed solution in the catheter circulate at a low speed and pass through the microbial settling chamber of the device. The large particles such as bacteria in the solution will settle in the settling chamber and be enriched on the surface of the metal LSPR sensor substrate at the bottom of the settling chamber.

[0053] S3, continuously collect the transmission spectrum of the LSPR sensor at different time points until the peak of the collected spectrum is obviously shifted, i.e. the bacteria in the original blood sample is detected, and the result is shown in Figure 3 The result shows that the obvious peak shift can be observed at 7h. This indicates that the device can directly detect the presence of microorganisms in the blood sample in the growth environment, and the proliferation and enrichment time is greatly shortened compared with the existing blood culture method.

[0054] Example 3: Selective rapid proliferation and detection method of a specific bacteria

[0055] This example is similar to example 2, except that in step S1, the specific antibody of Candida albicans is pre-modified on the surface of the LSPR sensor substrate, so that the sensor surface can specifically bind to Candida albicans.

[0056] In step S2, after the bacteria are enriched and cultured for a period of time, the fluid velocity of the detection device is increased to carry away the bacteria that are not bound to the surface of the LSPR sensor substrate, and the presence of Candida albicans in the solution is detected by the peak shift before and after.

[0057] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent flow table change made by using the content of the specification is also included in the patent protection scope of the present application.

Claims

1. A device for rapid proliferation and detection of microorganisms, characterized in that, It includes a light source (1), a peristaltic pump (2), a flexible tube (3), a microfluidic chip (4), a microbial sedimentation chamber (5), an optical sensing substrate (6), and a spectrometer (7). The microfluidic chip (4) includes a transparent cover plate (41) and a microfluidic channel layer (42). One end of the microfluidic channel layer (42) is provided with an inlet and the other end is provided with an outlet. A microfluidic straight channel is provided between the inlet and the outlet. The middle of the microfluidic straight channel is hollow and runs through the microfluidic channel layer (42). Its size matches the opening size of the microbial sedimentation chamber (5). The flexible tube (3) is connected end to end to the inlet and outlet of the microfluidic chip (4) through a peristaltic pump (2). The microbial sedimentation chamber (5) is located below the hollow structure of the microfluidic chip (4) and is connected to the microfluidic straight channel. The cross-section of the microbial sedimentation chamber (5) is much larger than the cross-section of the microfluidic straight channel. The optical sensing substrate (6) is placed at the bottom of the microbial sedimentation chamber (5). The bottom of the microbial sedimentation chamber (5) is a transparent structure. The light source (1) and the spectrometer (7) are located on the upper and lower sides of the microfluidic chip (4) and the microbial sedimentation chamber (5), respectively, and the light source (1), the microfluidic chip (4), the microbial sedimentation chamber (5) and the spectrometer (7) are on the same optical path; The optical sensing substrate (6) is a noble metal sensing array with local surface plasmon resonance effect that is sensitive to the surrounding refractive index; the microfluidic chip (4) is a PDMS microfluidic chip, including a PDMS cover plate and a PDMS microfluidic channel layer.

2. The apparatus according to claim 1, characterized in that, The hose (3) is a soft silicone tube.

3. The apparatus according to claim 1, characterized in that, The microbial sedimentation chamber (5) includes a chamber layer (51) of the same size as the microfluidic chip (4) and a transparent base plate (52); the chamber layer (51) is a substrate with a hollow structure in the middle, and the hollow structure is sealed to the transparent base plate (52) to form the microbial sedimentation chamber (5).

4. The apparatus according to claim 1, characterized in that, The noble metal sensing array is a three-dimensional gold nanopillar array, a gold nanopore array, or a gold nano mushroom head array.

5. The apparatus according to claim 1, characterized in that, The noble metal sensing array consists of an optically transparent substrate and a gold layer with a thickness of nanometers.

6. The apparatus according to claim 1, characterized in that, The spectrometer is an ultraviolet-visible spectrometer.

7. The apparatus according to claim 1, characterized in that, The surface of the optical sensing substrate (6) is modified with specific antibodies against the microorganism to be tested.

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