Renal function dry-type biochemical detection chip and use method thereof

Abstract

The invention belongs to the technical field of kidney disease detection, and particularly relates to a renal function dry-type biochemical detection chip and a use method thereof. The chip is combined with a dry biochemical reaction layer through a micro-fluidic technology, and can be used for synchronously detecting renal function markers such as urine creatinine, urea nitrogen and cystatin C. The chip adopts a multi-layer modular lamination design, and sequentially comprises a bottom plate, an electrode layer, a detection layer, a binding layer and a sample adding layer from bottom to top, and each layer is made of a high-molecular polymer with excellent biocompatibility, so that the detection process is not interfered, and the result is accurate and reliable. Sample flow is accurately controlled through the microfluidic channel, multi-index parallel detection is realized, and the detection efficiency is improved. A micro sensor is arranged in the chip, the reaction process is monitored in real time, and data accuracy is ensured. The system has the characteristics of portability and quick response, is suitable for clinical and family self-test, and provides a convenient and efficient solution for renal function monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of kidney disease detection, and in particular relates to a dry biochemical detection chip for kidney function and a method for using the same. Background Art

[0002] Accurate assessment of renal function is crucial for the early diagnosis and prevention of kidney disease. Urine creatinine (SCr), blood urea nitrogen (BUN), and cystatin C (cysC) are widely used in clinical practice to assess renal function. However, traditional detection methods typically require wet chemical analysis to measure these markers separately. This process is not only complex and time-consuming, but also susceptible to human interference, which can affect the accuracy of test results. Although existing dry biochemical detection chip technologies for renal function have enabled the simultaneous detection of multiple indicators to a certain extent, several challenges and issues remain in the specific detection of renal function markers. For example, when co-detecting multiple indicators, cross-contamination between different reaction systems, such as nonspecific binding of enzyme substrates and antibodies, can seriously affect detection accuracy. Furthermore, the stability of the electrochemiluminescence signal is often affected by uneven liquid flow rates, resulting in poor reproducibility of test results. Finally, current detection methods lack anti-interference design for the complex urine matrix, which leads to high background signals and further affects the sensitivity and specificity of detection.

[0003] Given these challenges, the development of a dry biochemical renal function test chip capable of simultaneously detecting three key markers: urine creatinine, urea nitrogen, and cystatin C, is urgent. This chip must not only possess robust anti-interference capabilities to ensure accurate test results, but also possess a highly integrated testing process to facilitate clinical application and improve detection efficiency. Such innovative technology will provide crucial support for the early diagnosis and treatment of kidney disease, possessing significant clinical application value and market potential. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a structurally optimized dry bipolar electrochemiluminescence dry biochemical detection chip for renal function. Through channel partitioning design, anti-interference coating modification and signal isolation technology, it can achieve simultaneous and accurate detection of urine creatinine, urea nitrogen and cystatin C, solving the problems of complex operation, signal interference and poor repeatability of traditional methods.

[0005] The present invention provides a dry biochemical detection chip for renal function for synchronously detecting urine creatinine, urea nitrogen and cystatin C. The chip comprises a laminated binding layer, a detection layer and an electrode layer.

[0006] A first labeling cavity, a second labeling cavity, and a third labeling cavity are formed inside the binding layer; a first labeling cavity is fixed in the first labeling cavity, and the first labeling cavity is used to bind to urine creatinine in the sample to be tested; a second labeling cavity is fixed in the second labeling cavity, and the second labeling cavity is used to bind to urea nitrogen in the sample to be tested; a third labeling cavity is fixed in the third labeling cavity, and the third labeling cavity is used to bind to cystatin C in the sample to be tested.

[0007] A first capture chamber, a second capture chamber, and a third capture chamber are formed inside the detection layer; the first capture chamber is connected to the first label chamber, the second capture chamber is connected to the second label chamber, and the third capture chamber is connected to the third label chamber. A first capture agent is fixed in the first capture chamber for capturing a first intermediate complex formed by urine creatinine and the first marker, a second capture agent is fixed in the second capture chamber for capturing a second intermediate complex formed by urea nitrogen and the second marker, and a third capture agent is fixed in the third capture chamber for capturing a third intermediate complex formed by cystatin C and the third marker.

[0008] A first detection anode, a second detection anode, a third detection anode and a common detection cathode are formed on the surface of the electrode layer. The first detection anode is attached to the first capture cavity and forms a first closed loop with the common detection cathode through an external driving power supply. The second detection anode is attached to the second capture cavity and forms a second closed loop with the common detection cathode through an external driving power supply. The third detection anode is attached to the second capture cavity and forms a third closed loop with the common detection cathode through an external driving power supply. The portion where the first capture cavity is attached to the first detection anode only allows light with a wavelength of 450 nm to pass through, the portion where the second capture cavity is attached to the second detection anode only allows light with a wavelength of 425 nm to pass through, and the portion where the third capture cavity is attached to the third detection anode only allows light with a wavelength of 620 nm to pass through.

[0009] Furthermore, the renal function dry biochemical detection chip also includes a sample loading layer. The sample loading layer has a first opening, a second opening, and a third opening defined on its top surface. The first opening extends into the sample loading layer and bifurcates to form a first loading channel, a second loading channel, and a third loading channel. The second opening extends into the sample loading layer and penetrates the sample loading layer to form a first buffer channel. The third opening extends into the sample loading layer and penetrates the sample loading layer to form a second buffer channel. The first buffer channel extends from the sample loading layer to the binding layer and bifurcates within the sample loading layer to form a first section, a second section, and a third section. The first labeling cavity communicates with the first loading channel via the first section, the second labeling cavity communicates with the second loading channel via the second section, and the third labeling cavity communicates with the third loading channel via the third section.

[0010] Furthermore, one end of the first marking cavity is connected to the first sample addition channel, and the other end is connected to the first section at an oblique angle, and the cross-sectional area of ​​the first marking cavity is larger than the cross-sectional area of ​​the first section.

[0011] Furthermore, one end of the second marking cavity is connected to the second sample addition channel, and the other end is connected to the second section at an oblique angle, and the cross-sectional area of ​​the second marking cavity is larger than the cross-sectional area of ​​the second section.

[0012] Furthermore, one end of the third marking cavity is connected to the third sample addition channel, and the other end is connected to the third section at an oblique angle, and the cross-sectional area of ​​the third marking cavity is larger than the cross-sectional area of ​​the third section.

[0013] Furthermore, the first marking cavity, the second marking cavity, and the third marking cavity are all filled with porous cellulose membranes.

[0014] Furthermore, the first marker is creatinine amide hydrolase-bipyridine ruthenium enzyme fixed on the surface of the porous cellulose membrane in the first labeling cavity through a weak chemical bond.

[0015] Furthermore, the second marker is urease-luminol enzyme fixed on the surface of the porous cellulose membrane in the second marking cavity through a weak chemical bond.

[0016] Furthermore, the third marker is an anti-CysC antibody-ruthenium complex fixed to the surface of the porous cellulose membrane in the third marker cavity through a weak chemical bond.

[0017] Furthermore, the second buffer channel passes through the thickness direction of the dry-type biochemical detection chip for renal function and branches in the detection layer to form a fourth section, a fifth section and a sixth section.

[0018] Furthermore, the first capture cavity has a first cross-section, a second cross-section and a first light-transmitting portion, the area of ​​the first cross-section is smaller than the area of ​​the second cross-section, the first marking cavity is connected to the first cross-section, the fourth section is connected to the second cross-section, the first light-transmitting portion is attached to the first detection anode, and the first light-transmitting portion only allows light with a wavelength of 450 nm to pass through.

[0019] Furthermore, the second capture cavity has a third section, a fourth section and a second light-transmitting portion, the area of ​​the third section is smaller than the area of ​​the fourth section, the second marking cavity is connected to the third section, the fifth section is connected to the fourth section, the second light-transmitting portion is attached to the second detection anode, and the second light-transmitting portion only allows light with a wavelength of 425 nm to pass through.

[0020] Furthermore, the third capture cavity has a fifth section, a sixth section and a third light-transmitting portion, the area of ​​the fifth section is smaller than the area of ​​the sixth section, the third marking cavity is connected to the fifth section, the sixth section is connected to the sixth section, the third light-transmitting portion is attached to the third detection anode, and the third light-transmitting portion only allows light with a wavelength of 620 nm to pass through.

[0021] Furthermore, the common detection cathode is equidistant from the first detection anode, the second detection anode, and the third detection anode on the surface of the electrode sheet.

[0022] Furthermore, the electrode layer also includes a first reporting channel, a second reporting channel, a third reporting channel, and a support channel formed inside the electrode layer. The first reporting channel, the second reporting channel, and the third reporting channel are all filled with conductive ink, and the support channel is filled with a recyclable electrolyte. The support channel is separated from the first reporting channel, the second reporting channel, and the third reporting channel. The first reporting channel is connected to the first detection anode side and extends into the electrode layer to connect to the driving negative electrode; the second reporting channel is connected to the second detection anode side and extends into the electrode layer to connect to the driving negative electrode; the third reporting channel is connected to the third detection anode side and extends into the electrode layer to connect to the driving negative electrode. The support channel is connected to the common detection cathode side and extends into the electrode layer to electrically connect to the driving positive electrode.

[0023] Furthermore, the dry-type biochemical detection chip for renal function also includes a bottom layer, which embeds the pins formed by the positive driving electrode and the negative driving electrode and forms an external connection port for electrical connection to an external device or an external power supply.

[0024] Furthermore, the renal function dry biochemical detection chip also includes a sample loading layer. One side of the sample loading layer is bonded to the binding layer, and the other side of the sample loading layer defines a first port, a second port, and a third port. The first port extends into the sample loading layer and bifurcates to form a first sample loading channel, a second sample loading channel, and a third sample loading channel, which extend through the thickness of the sample loading layer. The second port extends into the sample loading layer to form the first buffer channel, and the third port extends into the sample loading layer to form the second buffer channel.

[0025] The present invention also provides the use of the dry biochemical detection chip for renal function in preparing a kit or device for synchronously detecting urine creatinine, urea nitrogen, and cystatin C. Beneficial effects

[0026] The chip provided by the present invention has synchronous detection capabilities. Compared with existing multi-index detection chips and traditional methods, the chip of the present invention has a significant improvement in the number of single-detection indicators, and can simultaneously detect three indicators, including SCr, BUN and CysC, while the traditional method can only perform two tests, such as only protein or only nucleic acid, and requires single-indicator detection in batches. In addition, the chip of the present invention achieves spatial isolation and filter membrane technology through a channel cross-contamination rate of <1%, which is significantly better than the 5-10% contamination rate of traditional shared liquid paths. In terms of detection throughput, the chip of the present invention can process 3 samples at a time, and can reach 9 samples after expansion, while the traditional single sample / chip and single sample / batch methods are less efficient.

[0027] The chip realizes the simultaneous detection of three indicators (SCr, BUN, and CysC) through the design of three independent first detection anodes, second detection anodes, and third detection anodes and independent first reporting channels, second reporting channels, third reporting channels, and support channels. The single detection throughput can reach 3 samples (9 samples after expansion), which is more than 3 times the efficiency of traditional single-indicator fractionated detection.

[0028] The chip provided by the present invention features a silanized anti-adhesion coating (non-specific adsorption <1 ng / mm²) on the detection layer combined with a filter membrane as a double barrier, resulting in a cross-contamination rate of <1%, which is over 80% lower than that of existing shared channel chips. It is particularly suitable for complex samples such as hematuria and proteinuria.

[0029] The gold nanoparticle-modified electrodes in the detection layer of the chip provided by the present invention increase the reaction surface area. Combined with enzymatic signal amplification technology, the detection limit (LoD) reaches 0.1 mg / dL for urine creatinine, 0.5 mg / dL for urea nitrogen, and 0.01 mg / L for cystatin C, which is 10 times higher than that of traditional methods and meets the needs of early kidney injury detection.

[0030] The constant potential control (potential accuracy ±10mV) of the chip's independent electrode units combined with multi-wavelength spectroscopic detection (resolution ±5nm) provides a recovery rate of 97.8%-98.5% and a coefficient of variation (CV) of <3%, significantly superior to existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the planar structure of the chip of the present invention.

[0032] Figure 2 It is a schematic diagram of a three-dimensional structure including a binding layer 20, a detection layer 30 and an electrode layer 40.

[0033] Figure 1 、 Figure 2 The accompanying drawings are: Core layer 1000, sample loading layer 10, first port 12, first sample loading channel 121, second sample loading channel 122, third sample loading channel 123, second port 15, third port 16, first buffer channel 100, first section 101, second section 102, third section 103, second buffer channel 160, fourth section 161, fifth section 162, sixth section 163, binding layer 20, first labeling cavity 21, second labeling cavity 22, third labeling cavity 23, detection layer 30, first capture cavity 31, first cross section 311, second cross section 312, first light-transmitting portion 313, second capture chamber 32, third section 321, fourth section 322, second light-transmitting portion 323, third capture chamber 33, fifth section 331, sixth section 332, third light-transmitting portion 333, electrode layer 40, first detection anode 41, second detection anode 42, third detection anode 43, common bipolar electrode cathode 44, first reporting channel 451, second reporting channel 452, third reporting channel 453, supporting channel 46, driving positive electrode 47, driving negative electrode 48, bottom plate 50.

[0034] Figure 3 Statistical graphs of urine creatinine (SCr), urea nitrogen (BUN) and cystatin C (CCysC) detected using the chip of the present invention (A), the existing chip (B) and the traditional method (C).

[0035] Figure 4 The cross-reaction rates (CRRs) of simulated urine supplemented with hemoglobin (HGB), albumin (ALB) and uric acid (UA) were detected using the chip of the present invention (A) and the existing chip (B).

[0036] Figure 5 Radar graphs showing the detection of simulated urine with added hemoglobin (HGB), albumin (ALB) and uric acid (UA) using the chip of the present invention (A) and the conventional chip (B), respectively.

[0037] Figure 6 The recovery rates (RR) of urine from healthy individuals (NU) and urine from patients with kidney disease (Pro) were detected using the chip of the present invention (A), the existing chip (B), and the traditional method (C).

[0038] Figure 7 This is a Bland-Altman analysis chart showing the consistency of the test results using the chip of the present invention and the existing chip. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0040] Example 1: Chip Structure like Figure 1 As shown, the core layer utilizes a multi-layer modular stacked design, consisting, from bottom to top, of a base plate 50, an electrode layer 40, a detection layer 30, a binding layer 20, and a sample loading layer 10. This design clearly defines the functions of each component of the core layer, enabling efficient detection. The layers are secured by physical bonding, such as adhesives, and the channels between them are precisely aligned, ensuring fluid flow and signal conduction.

[0041] In some embodiments, the detection layer 30, the binding layer 20, and the sample loading layer 10 are integrated into one, and the electrode layer 40 and the base plate 50 are integrated into one, and the two integrated bodies are detachably connected, for example, by a magnetic structure or a modular snap connection, so as to facilitate the recycling of the detection layer 30, the binding layer 20, and the sample loading layer 10.

[0042] The sample loading layer 10 is located at the topmost layer of the core layer 1000. A first port 12, a second port 15, and a third port 16 are formed on the top surface of the sample loading layer 10. These ports 12, 15, and 16 are evenly distributed across the surface of the sample loading layer 10. The first port 12 is used for adding sample liquid. The first port 12 extends into the sample loading layer 10 and bifurcates to form a first loading channel 121, a second loading channel 122, and a third loading channel 123. These channels 121, 122, and 123 extend through the thickness of the sample loading layer 10. The second port 15 extends into the sample loading layer 10 to form a first buffer channel 100, and the third port 16 extends into the sample loading layer 10 to form a second buffer channel 160. A light shielding layer (aluminum foil coating) is added to the top surface of the sample loading layer 10, which can be removed during the regeneration phase.

[0043] The diameter of the first, second, and third ports 12, 15, and 16 are 2 mm, with a 5 mm spacing. The edges of the ports 12, 15, and 16 are hydrophobic to prevent liquid spillage. This design supports microinjection needles with an accuracy of ±1 μL, allowing for convenient and accurate addition of samples or buffers.

[0044] The first loading channel 121 , the second loading channel 122 and the third loading channel 123 have the same shape and an included angle of 60°, and each branch channel is 1.5 mm wide.

[0045] Binding layer 20 is attached to or formed beneath sample loading layer 10 and is also thin. A first labeling cavity 21, a second labeling cavity 22, and a third labeling cavity 23 are formed within binding layer 20. A first buffer channel 100 extends through the thickness of sample loading layer 10 and then continues into binding layer 20, bifurcating to form a first segment 101, a second segment 102, and a third segment 103.

[0046] One end of the first labeling cavity 21 is connected to the first sample loading channel 121 , and the other end is connected to the first section 101 at an oblique angle. The cross-sectional area of ​​the first labeling cavity 21 is larger than that of the first section 101 .

[0047] One end of the second marking cavity 22 is connected to the second sample loading channel 122 , and the other end is connected to the second section 102 at an oblique angle. The cross-sectional area of ​​the second marking cavity 21 is larger than that of the second section 102 .

[0048] One end of the third marking cavity 23 is connected to the third sample loading channel 123 , and the other end is connected to the third section 103 at an oblique angle. The cross-sectional area of ​​the third marking cavity 21 is larger than that of the third section 103 .

[0049] The first, second, and third marking chambers 21, 22, and 23 are all filled with porous cellulose membranes. Creatinine amidohydrolase-bipyridylruthenium enzyme is immobilized on the porous cellulose membrane surface in the first marking chamber 21 via weak chemical bonds (e.g., ionic bonds), with an enzyme activity >95%. Urease-luminolase is immobilized on the porous cellulose membrane surface in the second marking chamber 22 via weak chemical bonds (e.g., ionic bonds), with an enzyme activity >90%. An anti-CysC antibody-ruthenium complex is immobilized on the porous cellulose membrane surface in the third marking chamber 23 via weak chemical bonds (e.g., ionic bonds), with an antibody titer >1: .

[0050] The detection layer 30 is attached to or formed below the binding layer 20 and is also in the form of a thin layer. A first capture chamber 31, a second capture chamber 32, and a third capture chamber 33 are formed inside the detection layer 30.

[0051] The second buffer channel 160 penetrates along the thickness direction of the dry-type biochemical detection chip for renal function and branches at the detection layer 30 to form a fourth section 161 , a fifth section 162 and a sixth section 163 .

[0052] The first capture chamber 31 has a first cross-section 311, a second cross-section 312, and a first light-transmitting portion 313. The area of ​​the first cross-section 311 is smaller than that of the second cross-section 312. The first marking chamber 21 is connected to the first cross-section 311, and the fourth section 161 is connected to the second cross-section 312. The first light-transmitting portion 313 is in contact with the first detection anode 41 and only allows light with a wavelength of 450 nm to pass through. The first cross-section 311 is the cross-section at the smallest end of the fan-shaped first capture chamber 31. The second cross-section 312 is the cross-section at the largest end of the fan-shaped first capture chamber 31.

[0053] The second capture chamber 32 has a third cross-section 321, a fourth cross-section 322, and a second light-transmitting portion 323. The area of ​​the third cross-section 321 is smaller than that of the fourth cross-section 322. The second marking chamber 22 is connected to the third cross-section 321, and the fifth section 162 is connected to the fourth cross-section 322. The second light-transmitting portion 323 is in contact with the second detection anode 42 and only allows light with a wavelength of 425 nm to pass through. The third cross-section 321 is the cross-section at the smallest end of the fan-shaped second capture chamber 32. The fourth cross-section 322 is the cross-section at the largest end of the fan-shaped second capture chamber 32.

[0054] The third capture chamber 33 has a fifth cross-section 331, a sixth cross-section 332, and a third light-transmitting portion 333. The area of ​​the fifth cross-section 331 is smaller than that of the sixth cross-section 332. The third marking chamber 23 is connected to the fifth cross-section 331, and the sixth section 163 is connected to the sixth cross-section 332. The third light-transmitting portion 333 is in contact with the third detection anode 43 and only allows light of 620 nm to pass through. The fifth cross-section 331 is the cross-section at the smallest end of the fan-shaped portion of the third capture chamber 33. The sixth cross-section 332 is the cross-section at the largest end of the fan-shaped portion of the third capture chamber 33.

[0055] Anti-creatinine antibodies are immobilized on the inner wall of the first capture chamber 31 via an o-nitrobenzyl ether bond. Anti-urea nitrogen antibodies are immobilized on the inner wall of the second capture chamber 32 via an o-nitrobenzyl ether bond. Anti-cystatin C antibodies are immobilized on the inner wall of the third capture chamber 33 via an o-nitrobenzyl ether bond. The first, second, and third capture chambers 31, 32, and 33 are separated by a light-proof material forming a chip to prevent optical crosstalk.

[0056] When the marker brought into the binding layer 20 by the liquid in the first labeling chamber 21 flows into the first capture chamber 31 and combines with the anti-creatinine antibody on the inner wall of the first capture chamber 31 to form a "sandwich" structure marker of labeling antibody-creatinine-capture antibody, the buffer can be injected through the second buffer channel 160 to reversely flush the first capture chamber 31, so that the unbound substances are eluted by the buffer, thereby reducing the adsorption of nonspecific proteins.

[0057] Furthermore, the inner walls of these channels were modified with trimethylsilane coating by vapor deposition, with a contact angle of >110°, reducing the amount of nonspecific protein adsorption to <1ng / mm 2 The first capture channel 31, the second capture channel 32 and the third capture channel 33 have a volume of 50 μL and a depth of 0.2 mm.

[0058] The detection layer 30 is bonded to one surface of the electrode layer 40 , and the base plate 50 is bonded to the other surface thereof.

[0059] The electrode layer 40 is attached to one side of the detection layer 30 to form a first detection anode 41, a second detection anode 42, a third detection anode 43, and a common bipolar electrode cathode 44. The common bipolar electrode cathode 44 is equidistant from the first detection anode 41, the second detection anode 42, and the third detection anode 43 on the surface of the electrode layer 40.

[0060] The first detection anode 41 is attached to the first capture chamber 31 and forms a first closed loop with the common detection cathode 44 via an external driving power supply. The second detection anode 42 is attached to the second capture chamber 32 and forms a second closed loop with the common detection cathode 44 via an external driving power supply. The third detection anode 43 is attached to the third capture chamber 33 and forms a third closed loop with the common detection cathode 44 via an external driving power supply.

[0061] A first reporting channel 451, a second reporting channel 452, a third reporting channel 453, and a supporting channel 46 are formed within the electrode layer 40. The bottom surface of the electrode layer 40 is formed by a conductive layer with a positive driving electrode 47 and a negative driving electrode 48. The positive driving electrode 47 and the negative driving electrode 48 extend to both sides of the bottom surface of the electrode layer 40 to form conductive pins, which are then wrapped by the bottom layer 20 to facilitate connection to an external power source.

[0062] First, second, and third reporting channels 451, 452, and 453 are filled with conductive ink. Support channel 46 is filled with a recyclable KCl / NaCl mixed electrolyte and connected to an external ion exchange resin column (not shown) to remove electrochemical reaction byproducts in real time. Support channel 46 is isolated from first, second, and third reporting channels 451, 452, and 453.

[0063] The positive driving electrode 47 and the negative driving electrode 48 extend to both sides of the bottom surface of the electrode layer 40 to form conductive pins, which are wrapped by the bottom layer 20 to facilitate connection with an external power supply. The conductive layer is made of carbon nanotube-polyethylene glycol composite material with a resistivity of < The driving positive electrode 47 and the negative electrode 48 support bidirectional pulse voltage switching, and during regeneration, the electric field can be reversely applied to promote the complexes in the first capture chamber 31, the second capture chamber 32 and the third capture chamber 33 to separate from the electrode surface.

[0064] The first reporting channel 451 is connected to the side of the first detection anode 41 and extends into the interior of the electrode layer 40 to connect to the driving negative electrode 48, forming a closed current loop. The second reporting channel 452 is connected to the side of the second detection anode 42 and extends into the interior of the electrode layer 40 to connect to the driving negative electrode 48, forming a closed current loop. The third reporting channel 453 is connected to the side of the third detection anode 43 and extends into the interior of the electrode layer 40 to connect to the driving negative electrode 48, forming a closed current loop. The first reporting channel 451, the second reporting channel 452, and the third reporting channel 453 radiate in the plane direction of the electrode layer 40. The supporting channel 46 is connected to the side of the common detection cathode 44 and extends into the interior of the electrode layer 40 to be electrically connected to the driving positive electrode 47.

[0065] The bottom layer 50, made of PET, embeds the pins forming the positive and negative driving electrodes 47 and 48 and forms an external connection port for connecting to external devices or power sources. The bottom layer provides solid mechanical support for the entire core layer and connects to external devices through the electrode pins on its bottom surface, transmitting detection signals to external data collection equipment.

[0066] When an external power supply applies an 8V pulse voltage, electrons flow from the driving positive electrode 47 through the supporting channel 46 to the common detection cathode 44, where a reduction reaction occurs. Simultaneously, the first detection anode 41, the second detection anode 42, or the third detection anode 43 undergoes an oxidation reaction, releasing electrons that flow to the driving negative electrode 48 through the first reporting channel 451, the second reporting channel 452, and the third reporting channel 453, respectively, forming a closed current loop. The resistivity of the conductive layer inside the electrode layer 40 is < The impedance matching degree between the reporting channel and the supporting channel is >95%, ensuring the electronic transmission delay is <10ms. The laser cutting technology is used to process the channel inside the electrode layer 40, with an edge accuracy of ±5μm and an insulation resistance between the liquid and the circuit> , leakage current <1nA. The potential difference deviation of the first detection anode 41, the second detection anode 42, and the third detection anode 43 is less than 2%, supporting channel flow rate uniformity >98%, and achieving CV values ​​of less than 3% for all three indicators.

[0067] For example, a 20-μl urine sample is injected into the inlet of the sample layer. The sample is then diverted through the sample channel to the first branch channel, specifically for urine creatinine detection. Creatinine in the sample binds to creatinine amidohydrolase-ruthenium bipyridine in the first labeling chamber of the binding layer, forming a labeled species. Next, 50 μl of Tris-HCl buffer (pH 7.4) is injected through the first buffer channel, generating laminar shear force that dissociates the labeled species from the porous cellulose membrane and flows into the first capture chamber of the detection layer. Within the capture chamber, the labeled species binds to anti-creatinine antibodies immobilized on the inner wall of the channel, forming a "sandwich" structure. Buffer is then injected through the second buffer channel for backwashing, removing unbound species. Wastewater is then discharged through the first buffer channel. The labeled species immobilized in the capture chamber adheres to the surface of the first detection anode. When an 8-volt pulse voltage is applied from an external power source, an oxidation reaction occurs at the anode, emitting a 450-nanometer wavelength optical signal. This optical signal is captured by a CCD camera and converted into an electrical signal.

[0068] To regenerate the chip for reuse, the sample layer and light shielding layer are first removed. The capture chamber is then irradiated with UV light for 5 to 10 minutes using a fiber-optic UV light guide. This cleaves the o-nitrobenzyl ether bonds, releasing the sandwich complex. Next, 10 mM EDTA buffer (pH 9.0) is injected through the fourth port and circulated through the capture chamber at a flow rate of 50 μL / min using an ultrasonic vibrator. The complex is then discharged from the first buffer channel along with the waste solution. The external power supply is then switched to regeneration mode, and a 10 V pulse voltage (100 Hz frequency, 30 seconds duration) is applied. This utilizes the synergistic effects of electroosmotic flow and ultrasound to remove residual material. A 75% ethanol solution is then circulated for 10 minutes, followed by a rinse with deionized water until neutral. Finally, the impedance of each channel is measured using a test circuit integrated into the electrode layer to ensure a matching degree greater than 95% and a leakage current less than 1 nA. Simultaneously, the capture chamber of the detection layer is re-immobilized with capture reagent for the next use.

[0069] Example 2: Chip preparation process

[0070] 1. Material Preparation Sample loading layer (10), binding layer (20), detection layer (30): polytetrafluoroethylene (PTFE) sheets are used, with a thickness of 0.5 mm. The surface is modified with a zwitterionic polymer coating by vapor deposition, with a contact angle of >120° and a non-specific protein adsorption amount of <0.1 ng / mm².

[0071] Electrode layer (40): The substrate is a glass fiber epoxy resin board with a thickness of 1 mm, a titanium / platinum (Ti / Pt) conductive layer (thickness 200 nm) is sputtered on the surface, and the electrode pattern and internal channels are processed by laser cutting technology (edge ​​accuracy ±5 μm).

[0072] Bottom plate (50): PET material is selected, thickness is 2mm, and pin embedding groove is formed by injection molding process.

[0073] 2. Processing of each layer Sample layer processing: Use A laser engraver machine machined a sample injection port (2 mm diameter) and an internal channel (1.5 mm width, 0.3 mm depth) on the top surface of the PTFE sheet. The channel edges were plasma treated to render them hydrophobic (contact angle > 110°). A light-shielding layer of aluminum foil (50 μm thick) was applied to the top surface, and photolithography was used to define the light-transmitting areas.

[0074] Binding and detection layer fabrication: A labeling cavity (30 μL volume) and a capture cavity (50 μL volume, 0.2 mm depth) are formed within the PTFE sheet using a precision stamping process. An o-nitrobenzyl ether linker is attached to the cavity walls via plasma grafting. A wavelength-transmitting film with a thickness of 20 μm is heat-pressed onto the bottom surface of the detection layer's capture cavity to form the first, second, or third light-transmitting sections. The edges are sealed with UV-curable adhesive.

[0075] Electrode Layer Fabrication: A bipolar electrode array (anode size 2 mm × 2 mm, cathode size 5 mm × 5 mm) was formed on the surface of the conductive layer using a photolithography-electroplating process, with an electrode spacing of 10 mm. A reporting channel (0.3 mm diameter) and a supporting channel (0.5 mm diameter) were formed within the electrode layer using microchannel injection molding. The channels were filled with silver nanoparticle conductive ink and a KCl / NaCl electrolyte (concentration 1:1, pH 7.0), respectively.

[0076] 3. Chip Assembly Interlayer alignment: The sample layer, binding layer, and detection layer are stacked in sequence using a high-precision alignment platform (accuracy ±2μm) and fixed with UV-curable adhesive (transmittance >95%) to ensure that the interlayer channel deviation is <5μm.

[0077] Electrode connection: The detection layer and the electrode layer are aligned and connected through a magnetic attraction structure (NdFeB magnet, magnetic strength 50mT) to form a micro-gap electrochemical cell (gap 40-50μm).

[0078] Base plate packaging: The electrode layer pins are embedded in the base plate PET groove, and the overall packaging is achieved through a hot pressing process (temperature 150°C, pressure 5MPa). The final chip size is 25mm×25mm×2mm.

[0079] Example 3: Detection method and process

[0080] 1. Sample Pretreatment Urine sample: Use directly. If turbid, filter through a 0.45μm filter membrane (centrifugation conditions: 3000rpm, 5min). The detection temperature is controlled at 25±2℃.

[0081] 2. Testing steps 1. Chip pretreatment 20 μL of pretreatment solution (containing 0.1% Tween-20 and 50 mg / mL sucrose) was injected into the buffer channel through the second port 15 and the third port 16, and the channel was allowed to stand for 5 minutes to fully wet the channel, and then rinsed twice with deionized water.

[0082] 2. Sample addition reaction 20 μL of urine sample is injected into the first port 12 through a microinjection needle (accuracy ±1 μL). The liquid flows through the sample injection channel within 30 seconds under capillary action and enters the labeling cavity of the binding layer.

[0083] Urine creatinine reacts with creatinine amidohydrolase-bipyridylruthenium in the first labeling chamber 21 to form an enzymatic hydrolysis product-luminescent intermediate complex (reaction time 2 minutes). Urea nitrogen reacts with urease-luminol in the second labeling chamber 22 to produce an ammonia-luminol luminescent system (reaction time 1.5 minutes). Cystatin C reacts with an anti-CysC antibody-ruthenium complex in the third labeling chamber 23 to form an antigen-antibody complex (reaction time 2 minutes).

[0084] 50 μL of Tris-HCl buffer (pH 7.4, containing 0.1 M NaCl) was injected into the first buffer channel 100, and the complex was pushed through the filter membrane layer (molecular weight cutoff > 50 kDa) at a flow rate of 0.1 mL / min. The impurity removal rate was >98% in <1 min.

[0085] 3. Signal triggering and acquisition The chip was inserted into an electrochemiluminescence analyzer and an 8V pulse voltage (300ms duration, 1Hz frequency) was applied, causing oxidation reactions at each anode in sequence. Bipyridyl ruthenium oxidation produced a 450nm peak; luminol was oxidized by hydrogen peroxide under alkaline conditions to produce a 425nm peak; and the ruthenium complex was electrically excited to produce a 620nm peak. A CCD camera (50μm pixel resolution, 100ms exposure time) simultaneously acquired the luminescence intensity of the three channels. Background noise was subtracted using a built-in algorithm (based on baseline correction from a 365nm UV prescan) and concentrations were calculated using a standard curve (using a five-point calibration method with r² > 0.999). The entire process took 8 minutes.

[0086] 3. Chip Regeneration Process After sample analysis, the light shielding layer of the sample layer is removed. The capture chamber is then irradiated with a 365 nm UV light fiber (50 mW / cm²) for 5 to 10 minutes to cleave the o-nitrobenzyl ether bond, releasing the sandwich complex. Next, 10 mM EDTA buffer (pH 9.0) is injected through the fourth port, while ultrasonic vibration (40 kHz frequency, 30 W power) is activated. The capture chamber is flushed at a flow rate of 50 μL / min for 5 minutes, with the waste solution discharged through the first buffer channel. The external power supply is then switched to regeneration mode, applying a 10 V pulse voltage (100 Hz frequency, 50% duty cycle, 30 seconds duration) to remove residual material through the synergistic effect of electroosmotic flow and ultrasound. Finally, a 75% ethanol solution is flushed for 10 minutes, followed by a neutral rinse with deionized water. The impedance of each channel is measured using a built-in test circuit to ensure a matching degree greater than 95% and leakage current less than 1nA, completing the chemical disinfection and calibration steps. Simultaneously, the capture reagent is re-fixed into the capture chamber of the detection layer, and the chip is assembled for reuse.

[0087] 4. Detection Sensitivity Analysis To conduct the test, this test example first prepared standards of varying concentrations. The concentration range for urine creatinine was 0.01-1.0 mg / dL, the concentration range for urea nitrogen was 0.1-5.0 mg / dL, and the concentration range for cystatin C was 0.001-0.1 mg / L. During the test, each sample concentration was replicated 10 times, following the same "sample addition → signal trigger → data acquisition" procedure as in Example 3. For data processing, a standard curve of concentration versus luminescence intensity was plotted, and a linear regression equation was calculated, ensuring a correlation coefficient (R²) greater than 0.995. The limit of detection (LoD) was determined by adding three times the standard deviation to the mean luminescence intensity of a blank sample (ultrapure water). The specific calculation formula is: LoDblank = 3 × SD / Slope. According to these standards, the detection limit of urine creatinine should not exceed 0.1 mg / dL, the detection limit of urea nitrogen should not exceed 0.5 mg / dL, and the detection limit of cystatin C should not exceed 0.01 mg / L. These indicators meet the needs of early kidney injury detection.

[0088] Table 1

[0089] Table 2

[0090] As shown in Table 1, Table 2 and Figure 3As shown, the detection limits of urine creatinine, urea nitrogen and cystatin C of the present invention are significantly lower than those of existing electrochemiluminescence chips and traditional enzymatic / turbidimetric methods, showing higher sensitivity and better reproducibility.

[0091] (3) Detection specificity In order to prepare the interference sample, this test example added 100 mg / dL of hemoglobin to simulate hematuria, 50 g / L of albumin to simulate proteinuria, and 10 mg / dL of uric acid to simulate hyperuricemia to the blank urine matrix. Next, this test example performed five repeated tests on samples containing these interfering substances and blank samples without interfering substances to calculate the cross-reaction rate. The calculation formula for the cross-reaction rate is: cross-reaction rate = 100% × (interference sample detection value - blank sample detection value) / target standard detection value. In order to ensure that the detection signal is in the middle of the linear range, the concentration of the target standard is set to the clinical critical value, for example, 0.02 mg / L for cystatin C.

[0092] To verify the effectiveness of the anti-interference mechanism, this test case used scanning electron microscopy (SEM) to observe nonspecific adsorption on the surface of the detection layer channel. Protein adsorption was required to be less than 1 ng / mm², which was verified using the BCA protein quantification method. Furthermore, this test case used high-performance liquid chromatography (HPLC) to analyze sample composition before and after the filter membrane to verify that the retention rate of large molecular impurities exceeded 98%. The final judgment criteria were that the cross-reactivity rates for hemoglobin, albumin, and uric acid should all be less than 1%, and this should be reduced by more than 80% compared to existing chips, demonstrating the effectiveness of the dual barrier of the anti-interference coating and filter membrane.

[0093] Table 3

[0094] As shown in Table 2, Figure 4 and Figure 5 As shown, the cross-reactivity rates of hemoglobin, albumin, and uric acid in the present invention are significantly lower than those of existing chips, significantly improving anti-interference capabilities and ensuring the accuracy and reliability of test results. The silanized anti-adhesion coating on the test chip reduces nonspecific protein adsorption to less than 1ng / mm². Combined with the filter membrane's ability to intercept macromolecular impurities, the cross-reactivity rate is reduced by over 80% compared to existing chips, making it particularly suitable for complex clinical samples such as hematuria and proteinuria.

[0095] Detection accuracy To prepare the standards and spiked samples, this test case first collected urine samples from healthy individuals to ensure they were free of visible impurities. The urine was then filtered through a 0.45μm filter membrane and liquid chromatography-mass spectrometry (LC-MS) was used to confirm the absence of the target analytes. Specifically, these samples contained urine creatinine levels below 0.05mg / dL, urea nitrogen below 0.3mg / dL, and cystatin C below 0.005mg / L.

[0096] Next, this test case added three different concentrations of standard substances, high, medium, and low, to these blank urine samples to cover the range of clinical testing. The specific added concentrations are as follows: Urine creatinine: low concentration is 0.5 mg / dL, medium concentration is 5.0 mg / dL, and high concentration is 20.0 mg / dL; Urea nitrogen: low concentration is 2.0 mg / dL, medium concentration is 10.0 mg / dL, and high concentration is 50.0 mg / dL; Cystatin C: low concentration is 0.02 mg / L (close to the clinical critical value), medium concentration is 0.1 mg / L, and high concentration is 1.0 mg / L.

[0097] Six parallel samples were prepared for each concentration in this test example.

[0098] During the detection process, this test example tested the spiked sample according to the steps of Example 3, recorded the luminescence intensity of each indicator, and then calculated the measured concentration.

[0099] In terms of data processing, this test case calculated the recovery using the formula: measured concentration divided by spiked concentration, then multiplied by 100%. The recovery was required to be within the range of 95%-105% (with a clinically acceptable error of ±5%). The average recovery and standard deviation (SD) for six samples were calculated. Furthermore, the recovery of spiked urine samples was compared with that of standard solutions of equal concentration to assess matrix effects (ME). Ideally, the ME should be close to 100%, with an acceptable deviation of ±8%. The coefficient of variation (CV) of the measured values ​​for spiked samples of equal concentration was also calculated, with a requirement of less than 3% to verify the repeatability of the test.

[0100] In terms of clinical sample comparison, this test case collected urine samples from 30 patients with clinically confirmed kidney disease approved by the hospital ethics committee and tested them using the chip of the present invention and traditional methods (urine creatinine: picric acid method; urea nitrogen: enzyme-coupled rate method; cystatin C: immunoturbidimetric method). This test case used the Bland-Altman analysis method ( Figure 7) evaluated the consistency of the present invention and the traditional method, and calculated the bias (Bias) and 95% consistency limits (LoA), requiring the bias to be less than ±5% and the LoA range to be between -10% and +10%.

[0101] Finally, according to the judgment criteria, the average recovery rate of spiked samples was 97.8%-98.5%, the CV was less than 3%, and the matrix effect ME was between 92%-108%; a comparison of clinical samples showed that the correlation coefficient r of the detection results of the chip of the present invention and the traditional method was greater than 0.99, and the Bland-Altman bias was within an acceptable range, proving that the detection accuracy of the chip of the present invention is superior to that of existing chips (the recovery rate of existing chips is between 89.7%-92.3%, and the CV is greater than 4%).

[0102] Table 4

[0103] As shown in Table 3 and Figure 6 As shown, the chip's recovery rates in urine samples from both healthy individuals and kidney patients were significantly higher than those of existing chips, and closer to traditional methods, further validating its high accuracy and reliability. Furthermore, the chip's constant potential control accuracy of ±10mV through independent electrode units ensures luminescence signal stability. Combined with a multi-wavelength spectroscopic detection resolution of ±5nm, it eliminates spectral overlap interference, resulting in an 8% improvement in accuracy compared to existing single-wavelength detection chips.

[0104] This invention overcomes bottlenecks such as cross-contamination and insufficient sensitivity in traditional multi-parameter detection through its unique structural design of "spatial isolation electrodes + anti-interference functional layer + multi-signal spectroscopic detection." Compared with existing technologies, the chip significantly improves key indicators such as simultaneous detection capability, sensitivity, specificity, and accuracy. It is particularly suitable for early screening and rapid bedside diagnosis of renal impairment in clinical practice, and has significant medical application value and industrial prospects.

[0105] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. A dry biochemical detection chip for renal function for simultaneous detection of urine creatinine, urea nitrogen, and cystatin C, characterized in that: include: A binding layer, wherein a first marking cavity, a second marking cavity, and a third marking cavity are formed inside the binding layer; A first marker is fixed in the first labeling cavity, and the first marker is used to bind to urine creatinine in the sample to be tested; a second marker is fixed in the second labeling cavity, and the second marker is used to bind to urea nitrogen in the sample to be tested; a third marker is fixed in the third labeling cavity, and the third marker is used to bind to cystatin C in the sample to be tested; A detection layer, wherein a first capture cavity, a second capture cavity, and a third capture cavity are formed inside the detection layer; The first capture chamber is connected to the first label chamber, the second capture chamber is connected to the second label chamber, and the third capture chamber is connected to the third label chamber. A first capture agent is fixed in the first capture chamber for capturing a first intermediate complex formed by urine creatinine and the first marker, a second capture agent is fixed in the second capture chamber for capturing a second intermediate complex formed by urea nitrogen and the second marker, and a third capture agent is fixed in the third capture chamber for capturing a third intermediate complex formed by cystatin C and the third marker. An electrode layer is provided, wherein a first detection anode, a second detection anode, a third detection anode and a common detection cathode are formed on the surface of the electrode layer, the first detection anode is adhered to the first capture cavity and forms a first closed loop with the common detection cathode through an external driving power supply, the second detection anode is adhered to the second capture cavity and forms a second closed loop with the common detection cathode through an external driving power supply, the third detection anode is adhered to the second capture cavity and forms a third closed loop with the common detection cathode through an external driving power supply, wherein the portion where the first capture cavity is adhered to the first detection anode only allows light with a wavelength of 450 nm to pass through, the portion where the second capture cavity is adhered to the second detection anode only allows light with a wavelength of 425 nm to pass through, and the portion where the third capture cavity is adhered to the third detection anode only allows light with a wavelength of 620 nm to pass through.

2. The dry biochemical detection chip for renal function according to claim 1, characterized in that: Also includes: A sample loading layer, wherein a first opening, a second opening, and a third opening are formed on a top surface of the sample loading layer. The first opening extends into the sample loading layer and bifurcates to form a first sample loading channel, a second sample loading channel, and a third sample loading channel. The second opening extends into the sample loading layer and penetrates the sample loading layer to form a first buffer channel. The third opening extends into the sample loading layer and penetrates the sample loading layer to form a second buffer channel. The first buffer channel extends from the sample loading layer to the binding layer and bifurcates into a first section, a second section, and a third section within the sample loading layer. The first labeling cavity is connected to the first sample loading channel through the first section, the second labeling cavity is connected to the second sample loading channel through the second section, and the third labeling cavity is connected to the third sample loading channel through the third section.

3. The dry biochemical detection chip for renal function according to claim 1, characterized in that: One end of the first labeling cavity is connected to the first sample addition channel, and the other end is connected to the first section at an oblique angle, and the cross-sectional area of ​​the first labeling cavity is larger than the cross-sectional area of ​​the first section; One end of the second labeling cavity is connected to the second sample addition channel, and the other end is connected to the second section at an oblique angle, and the cross-sectional area of ​​the second labeling cavity is larger than the cross-sectional area of ​​the second section; One end of the third marking cavity is connected to the third sample addition channel, and the other end is connected to the third section at an oblique angle. The cross-sectional area of ​​the third marking cavity is larger than that of the third section.

4. The dry biochemical detection chip for renal function according to claim 1, characterized in that: The first marking cavity, the second marking cavity, and the third marking cavity are all filled with porous cellulose membranes; The first marker is creatinine amide hydrolase-bipyridine ruthenium enzyme fixed on the surface of the porous cellulose membrane in the first labeling cavity through a weak chemical bond; The second marker is urease-luminol enzyme fixed on the surface of the porous cellulose membrane in the second marker cavity through a weak chemical bond; The third marker is an anti-CysC antibody-ruthenium complex fixed on the surface of the porous cellulose membrane in the third marker cavity through a weak chemical bond.

5. The dry biochemical detection chip for renal function according to claim 1, characterized in that: The second buffer channel runs through the thickness direction of the renal function dry biochemical detection chip and branches in the detection layer to form a fourth section, a fifth section and a sixth section; The first capture cavity has a first cross-section, a second cross-section, and a first light-transmitting portion. The area of ​​the first cross-section is smaller than the area of ​​the second cross-section. The first labeling cavity is connected to the first cross-section. The fourth section is connected to the second cross-section. The first light-transmitting portion is attached to the first detection anode. The first light-transmitting portion only allows light with a wavelength of 450 nm to pass through. The second capture cavity has a third cross-section, a fourth cross-section, and a second light-transmitting portion, the third cross-section is smaller than the fourth cross-section, the second labeling cavity is connected to the third cross-section, the fifth section is connected to the fourth cross-section, the second light-transmitting portion is attached to the second detection anode, and the second light-transmitting portion only allows light with a wavelength of 425 nm to pass through; The third capture cavity has a fifth section, a sixth section and a third light-transmitting portion, the area of ​​the fifth section is smaller than the area of ​​the sixth section, the third marking cavity is connected to the fifth section, the sixth section is connected to the sixth section, the third light-transmitting portion is attached to the third detection anode, and the third light-transmitting portion only allows light with a wavelength of 620 nm to pass through.

6. The dry biochemical detection chip for renal function according to claim 1, characterized in that: The common detection cathode is equidistant from the first detection anode, the second detection anode, and the third detection anode on the surface of the electrode sheet.

7. The dry biochemical detection chip for renal function according to claim 1, characterized in that: The electrode layer further comprises: A first reporting channel, a second reporting channel, a third reporting channel, and a supporting channel are formed within the electrode layer, wherein the first reporting channel, the second reporting channel, and the third reporting channel are all filled with conductive ink, and the supporting channel is filled with a recyclable electrolyte, and the supporting channel is separated from the first reporting channel, the second reporting channel, and the third reporting channel; and A driving positive electrode and a driving negative electrode formed on a side of the electrode layer away from the detection layer; The first reporting channel is connected to the first detection anode side and extends into the electrode layer to connect to the negative driving electrode; the second reporting channel is connected to the second detection anode side and extends into the electrode layer to connect to the negative driving electrode; the third reporting channel is connected to the third detection anode side and extends into the electrode layer to connect to the negative driving electrode; The supporting channel is connected to the common detection cathode side and extends into the electrode layer until it is electrically connected to the driving positive electrode.

8. The dry biochemical detection chip for renal function according to claim 1, characterized in that: The invention also includes a bottom layer, which buries the pins formed by the driving positive electrode and the driving negative electrode and forms an external connection port to facilitate electrical connection to an external device or an external power supply.

9. The dry biochemical detection chip for renal function according to claim 1, characterized in that: It also includes a sample adding layer, one side of the sample adding layer is attached to the binding layer, and the other side of the sample adding layer is provided with a first port, a second port, and a third port; The first opening extends into the sample loading layer and bifurcates to form a first sample loading channel, a second sample loading channel, and a third sample loading channel, and the first sample loading channel, the second sample loading channel, and the third sample loading channel penetrate the sample loading layer in a thickness direction; The second port penetrates into the sample loading layer to form the first buffer channel; The third port penetrates into the sample loading layer to form the second buffer channel.

10. Use of the dry biochemical detection chip for renal function according to any one of claims 1 to 9 in the preparation of a kit or device for the simultaneous detection of urine creatinine, urea nitrogen and cystatin C.

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