Patent Publication Number: US-2023138856-A1

Title: Capillary array electrophoresis-chemiluminescence detection coupled system

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of priority from Chinese Patent Application No. 202111637304.5, filed on Dec. 30, 2021. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein by reference in its entirety. 
     TECHNICAL FIELD 
     This application relates to capillary array electrophoresis (CAE) analysis, and more particularity to a CAE-chemiluminescence detection coupled system for bioanalysis, clinical diagnosis, environmental monitoring, and food safety analysis. 
     BACKGROUND 
     Capillary electrophoresis (CE) is an efficient separation and analysis method based on the difference in charge-mass ratio of individual components using capillary as the electrolyte support and a high-voltage electric field as the driving force. The CE has advantages of high separation efficiency, rapid analysis and less sample and reagent consumption, and thus has been widely used in bioanalysis, clinical diagnosis, environmental monitoring, and food safety analysis. Compared to the traditional single-capillary electrophoresis, capillary array electrophoresis (CAE) enables the high-throughput analysis by means of a capillary array, and thus has been widely used in genetic analysis and clinical diagnosis. 
     At present, the CAE instruments mainly adopt a laser fluorescence detector and an ultraviolet-visible (UV-vis) detector. The laser fluorescence detector is a selective detector with high sensitivity, and has been widely used for genetic sequencing and analysis. The laser fluorescence detector can operate under an imaging mode and a scanning mode. Regarding the imaging mode, a parallel beam of light is emitted to capillaries arrayed in parallel, and the fluorescence emitted by the capillary array is collected by a plane or linear array detector (e.g., charge coupled device (CCD) and diode array detector (DAD)). Whereas, there is fluorescence signal interference between adjacent capillaries. Regarding the scanning mode, a focused laser beam is employed to scan the capillaries one by one, and the fluorescence signal from individual capillaries is collected by a single photon detector such as photomultiplier tube, eliminating the fluorescence and scattering light signal interference between adjacent capillaries. Unfortunately, under the scanning mode, the laser beam or capillary array is often moved mechanically, which leads to poor data collection efficiency. The UV-vis detector has strong versatility, and generally adopts an imaging mode and a fiber optic detection mode. Similar to the laser fluorescence detector, there is also scattering light signal interference in the UV-vis detector under the imaging mode. Regarding the fiber optic detection mode, an excitation fiber (incident light) and a collection fiber are employed to collect an optical absorption signal of each capillary, effectively avoiding the scattering light signal interference between adjacent capillaries. Nevertheless, this mode struggles with low throughput, and the sensitivity of the UV detector is relatively poor, so that the UV-vis detector fails to realize the analysis of low-concentration components. Moreover, the existing CAE detectors (laser fluorescence and UV-vis detectors) all adopt a light source as an excitation source for the sample signal, the background signal generated by which will significantly reduce the sensitivity of the detection system. 
     SUMMARY 
     An object of this disclosure is to provide a CAE-chemiluminescence (CL) detection coupled system, which is free of an excitation source, and has simplified CAE structure and reduced costs. The CAE-CL detection coupled system provided herein has excellent sensitivity and high detection throughput, and can be used in bioanalysis, clinical diagnosis, environmental monitoring and food safety analysis. 
     Technical solutions of this application are described as follows. 
     This application provides a CAE-CL detection coupled system, comprising: a power supply with a voltage of 5-30 kV; 
     a capillary array; 
     an array channel CL reaction tank; 
     an electrophoresis anode tank (CAE electrophoresis buffer tank); 
     an electrophoresis cathode tank (CAE electrophoresis buffer tank); 
     an imaging lens; 
     a plane array or linear array detector; 
     a data acquisition and processing unit; and 
     a computer; 
     wherein the electrophoresis anode tank is connected to an anode of the power supply; an outlet end of the capillary array is connected to the array channel CL reaction tank; and the array channel CL reaction tank is connected to the electrophoresis cathode tank, such that the capillary array is further connected to the CAE detection tank. A sample flows out from the outlet end of the capillary array, and then reacts with a chemical reagent to generate CL. The CL is collected by the imaging lens, and the plane array or linear array detector converts an optical signal into an electrical signal. The electrical signal is subjected to the data acquisition and processing unit to obtain a capillary array electrophoretogram. 
     In some embodiments, the array channel CL reaction tank is provided with a capillary array inlet, a chemiluminescent reagent input port and an output port. The array channel CL reaction tank is provided with a plurality of microchannels. The number of the plurality of microchannels is 2-384. A diameter of each microchannel of the plurality of microchannels is 200-5000 μm. Each capillary of the capillary array is located in a corresponding microchannel. The chemiluminescent reagent generates a sheath flow around each capillary driven by a delivery pump. When a separated component flows out of the capillary array, it is subjected to CL reaction with the chemiluminescent reagent. 
     In some embodiments, a chemiluminescent reagent delivery unit comprises a chemiluminescent substrate tank, an oxidant tank, the delivery pump and a mixer. The mixer is configured to mix a chemiluminescent substrate and an oxidant according to a preset ratio and then convey the mixture to the array channel CL reaction tank. The flow rate and mixing ratio are controlled by the computer. 
     In some embodiments, a chemiluminescent reagent comprises the chemiluminescent substrate and the oxidant. The chemiluminescent substrate is selected from the group consisting of luminol, luminol derivatives (such as isoluminol) and peroxyoxalate esters compounds; and the oxidant is selected from the group consisting of hydrogen peroxide, sodium peroxide, potassium peroxide, permanganate, periodate, hypochlorite, dichromate, ammonium persulfate and ceric sulfate. 
     In some embodiments, the CAE detection tank comprises a plurality of optical detection windows arranged at a side of the capillary array, an electrophoresis buffer inlet, a waste liquid outlet and a metal electrode (platinum electrode); the array channel CL reaction tank is connected to the electrophoresis cathode tank; and the high-voltage power supply is communicated with the electrophoresis cathode tank and the electrophoresis anode tank. 
     In some embodiments, a multi-channel detection unit comprises the imaging lens and the plane array or linear array detector, wherein the plane array detector is CCD or a complementary metal oxide semiconductor (CMOS), and the linear array detector is DAD. A photosensitive surface of the array detector faces towards an axial direction of the outlet end of the capillary array (namely the axial detection mode), or faces towards the plurality of optical detection windows near the outlet end of the capillary array (namely the lateral detection mode). In the lateral direction detection mode, a length of each of the plurality of optical detection windows is 1-10 mm; and a distance between the imaging lens and the outlet end of the capillary array (or the optical detection window) is a focal length of the imaging lens. 
     In some embodiments, the data acquisition and processing unit comprises a data acquisition card and a data processing software; the data acquisition card is connected to the plane array or linear array detector, and is configured to collect a signal of individual channels of the plane array or linear array detector in real time, record a capillary electrophoretogram, and calculate a peak area, peak height and migration time of the separated component in individual channels of the plane array or linear array detector. The data acquisition card is connected to the computer to perform automatic data acquisition and processing. 
     Compared to the prior art, the present disclosure has the following beneficial effects. 
     (1) The CAE-CL detection coupled system provided herein is free of a light source. The capillaries can be arranged in a two-dimensional (2D) array (plane array), which remarkably improves the throughput of the capillary array (the number of the capillaries). 
     (2) The CAE-CL detection coupled system provided herein eliminates the mechanical scanning, and thus can achieve the fast data acquisition and rapid separation and detection. 
     (3) Regarding the CAE-CL detection coupled system provided herein, the light source is not required. A plane array detector is adopted at the outlet end of the capillary array for detection, such that a signal interference between adjacent capillaries is eliminated, and there is no background signal caused by Rayleigh scattering, Raman scattering and solvent fluorescence impurities, contributing to excellent sensitivity. Therefore, the CAE-CL detection coupled system provided herein can be applied in bioanalysis, clinical diagnostics, environmental monitoring, and food safety analysis. 
     (4) The CAE-CL detection coupled system provided herein has high sensitivity, and is thus suitable for the CAE detection of low-concentration components. 
     (5) Due to the absence of a light source, the system provided herein has a simplified structure and reduced costs. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    schematically shows a structure of a CAE-CL detection coupled system according to an embodiment of the disclosure; 
         FIG.  2    is a side view of an array channel CL reaction tank under an axial detection mode; 
         FIG.  3    is a sectional view of a microchannel array in the array channel CL reaction tank under the axial detection mode; 
         FIG.  4    is a side view of the array channel CL reaction tank under a lateral detection mode; 
         FIG.  5    is a sectional view of the microchannel array in the array channel CL reaction tank under the lateral detection mode; 
         FIG.  6    is a capillary array electrophoretogram of horseradish peroxidase (HRP) under the axial detection mode; and 
         FIG.  7    is a capillary array electrophoretogram of HRP under the lateral detection mode. 
     
    
    
     In the drawings:  1 , platinum anode;  2 , platinum cathode;  3 , CAE sample tank;  4 , capillary array;  5 , high-voltage power supply;  6 , array channel CL reaction tank;  7 , CAE detection tank;  8 , chemiluminescent substrate tank;  9 , oxidant tank;  10 , mixer (including a delivery pump);  11 , electrophoresis buffer storage tank;  12 , imaging lens;  13 , plane array detector;  14 , waste liquid tank;  15 , data acquisition and processing unit; and  16 , computer. 
     DETAILED DESCRIPTION OF EMBODIMENTS 
     Embodiment 1 
     The CAE detection of HRP was conducted under an axial detection mode of a CAE-CL detection coupled system (shown in  FIGS.  1 - 3   ) and the capillary array electrophoretogram was shown in  FIG.  6   , where the capillary array consisted of five capillaries is 5; and the sample was 1.0×10 −8  mol/L HRP. Referring to  FIG.  6   , the sample peak was observed in the 2 nd -the 5 th  capillaries. The 1 st  capillary without the sample was free of the sample peak, indicating that its detection was not interfered by other capillaries (such as the 4 th  capillary and the 5 th  capillary). 
     The capillaries were made of quartz, each having an inner diameter of 50 μm and a length of 30 cm. The electrophoresis buffer was 3.7 mmol/L sodium borate solution (pH=10.2). The CL reagent was prepared by 50 mmol/L NaHCO 3  (pH=9.0), 7.5×10 −3  mol/L H 2 O 2 , 7.5×10 −4  mol/L luminol and 1.25×10 −3  mol/L ethylenediaminetetraacetic acid (EDTA). The electrophoresis was conducted at 15 kV. As shown in  FIG.  1   , the sample was introduced to the capillary array  4  from the platinum anode  1 , and then migrated to the platinum cathode  2  under the high electric field provided by the high-voltage power supply  5 . After electrophoresis time, the separated sample flowed out of the outlet end of the capillary and met with chemiluminescent reagents (fed from the chemiluminescent substrate tank  8  and the oxidant  9 ) in the array channel CL reaction tank  6 . The generated chemiluminescent signal was captured by an imaging lens  12  and a plane array detector  13  (CCD), and was processed with data acquisition and processing unit  15 . In this case, the photosensitive surface of CCD faced towards the axial direction of the outlet end of the capillary array. The capillary array electrophoretogram was shown in  FIG.  6    and was output by a computer  16 . 
     Embodiment 2 
     The CAE detection of HRP was conducted under a lateral detection mode of a CAE-CL detection coupled system (shown in  FIGS.  1  and  4 - 5   ), and the capillary array electrophoretogram was shown in  FIG.  7   , where the capillary array consisted of five capillaries, and the sample was 1.0×10 −8  mol/L HRP. Referring to  FIG.  7   , the sample peak was observed in the 1 st  capillary and the 3rd-5 th  capillaries. The 2 nd  capillary without sample loading was free of the sample peak, indicating that other capillaries (such as the 1 st  capillary and the 3 rd -5 th  capillaries) had no interference with its detection. 
     The capillaries were made of quartz, each having an inner diameter of 50 μm and a length of 36 cm. The electrophoresis buffer was 3.7 mmol/L sodium borate solution (pH=10.2). The CL reagent was prepared by 50 mmol/L NaHCO 3  (pH=9.0), 7.5×10 −3  mol/L H 2 O 2 , 7.5×10 −4  mol/L luminol and 1.25×10 −4  mol/L EDTA. The electrophoresis was performed at 10 kV. In this case, the photosensitive surface of CCD faced towards the lateral direction of the outlet end of the capillary array.