A novel bio-microcircuit for bio-assays

Abstract

In this study, a novel micro-system made of a fluid circuit board and various functional components, inspired by integrated microcircuit techniques, was developed. The circuit board was fabricated on a thermoplastic sheet using two-dimensional (2D) cutting technology. The functional components, including mixers, dilutors, reactors, pumps, retreaters, and detectors, were fabricated via paper (or membrane) cutting/folding and integrated into the system, similar to the way that electronic components are integrated into a micro-integrated circuit board. This system, validated through the rapid detection of human immunodeficiency virus (HIV) and the starch catabolism process, could potentially be another microfluidic system for analytical science.

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A microcircuit is a type of micro-integrated electronic system that combines transistors, diodes, resistors, capacitors and inductors, in which a driving force from a voltage applied to electrodes is transported into the circuit to activate the microelectronic components, performing a series of electrical functions. This microcircuit system could rationally be extended into the biological sciences.^1,2 In this study, the novel concept of a micro-system is developed, which transports, manipulates and processes bio-molecules (amino acids, sugars, fatty acids, nucleotide acids, inorganic salts, water and even bio-electrodes) through vessels, micro-channels, and certain types of fiber substrates, and then performs various biochemical reactions and completes fully integrated bio-functions.³ We called this kind of micro-system a bio-microcircuit (or another type of microfluidics).

There are some other techniques for the fabrication of micro-channels, for example, photolithography,^4,5 plotting,⁶ plasma treatment,^7,8 ink jet etching/printing^9,10 wax printing,^11,12 etc., while in this work, we made use of 2-D cutting techniques to construct the micro-channels and transport the liquid using capillary force. Most traditional microfluidics, based on typical fabrication methods, realize their functions via special planar and homogenized micro-channel structures. However, our proposed system (bio-microcircuit) could be much more flexible and extendable.

This type of system is already found in nature in the blood circulatory system (where blood cells and other substances are transported through the blood vessels by the force of the heart's movement to maintain life movements),¹³ the nerve conduction system (where bio-electrodes are transported along nerve axons by electrical stimulation to complete a series of neutral responses),¹⁴ and the plant transportation system (where water and nutrients are transported through plant vessels by the pulling force of transpiration).^15,16

In this project, we imitate microelectronics and life biology, and describe a novel micro-system and its construction using cutting/folding technology, and demonstrate its application in the rapid detection of human immunodeficiency virus (HIV) and starch catabolism.

Results and discussion

Schematics of this novel micro-system

The micro-system was composed of a circuit board and various types of functional components. The circuit board was a flat substrate on which either vessels or channels (instead of the electrical wires in a microcircuit system) were constructed and used to transport bio-substances. The functional components were fabricated and assembled in a certain way on the circuit board to realize a particular biochemical function.

Two-dimensional (2D) cutting technology was applied to construct the circuit board (Fig. 1).^17,18 This technology utilized a cutting machine that incorporated a shape blade in place of the traditional printing/plotting pen in desktop printers/plotters. The blade rotated freely on a turret, enabling the precise cutting of various patterns on a flat substrate using computerized X-Y knife cutter software (Circuit Expression® 2, Provo Craft & Novelty, Inc., USA).¹⁹ This flat substrate could be thermoplastic plates, paper, polymers, membranes, or other porous materials in which different channel/vessel depths could be constructed and patterned by adjusting the blade angle and downward force. In this study, a thermoplastic sheet was used. The depth and width of the micro channels were approximately 300 μm and 250 μm, respectively. The micro-channels generated were embedded in the thermoplastic plates, and could be sealed using tape if necessary, to transport liquid.

Fig. 1 Schematic illustration of the fabrication of this novel system. A computer-directed X-Y knife cutting machine is used to construct the circuit board and various types of functional components.

Ink of a red color was applied to demonstrate that fluid could be transported in this micro-system. The pattern of micro-channels on the thermoplastic sheet could also be sealed with plastic tape and then the fluid could be driven using a pumping force.

Fabrication of functional components

In addition to the circuit board, various types of functional biological components were constructed using the cutting and folding technology, including sample inlets, sample pre-treaters, mixers, separators, dilutors, reactors and detectors. These analogues to electrical components (resistors, capacitors and transistors) can be used to carry out various functions in this novel micro-system (Fig. 2). The sample inlet was constructed directly by cutting a spiral-like micro-channel in a thermoplastic sheet, in which the sample loading depended primarily on the dimensions of the spiral. As illustrated in Fig. 2a, different sample volumes of 2 μL (red ink), 4 μL (yellow ink), 6 μL (green ink) and 10 μL (blue ink) were loaded in a 2 mm, 4 mm, 6 mm, and 10 mm spiral, respectively, and were transported into the micro-system using capillary force. The samples were pretreated with a square piece of cellulose paper coated with a pretreatment reagent. As shown in Fig. 2b, a sample of green ink was transported from the input and became dark within 15 s, after processing with the pretreatment. Other pretreatments, such as pH/ionic strength adjustment, impurity removal and interference shielding, could all easily be performed in a similar way using this component. Various mixers were designed in this study, including ellipsoid, sausage-like and three/four-member ring-like mixers. The four-member ring-like mixer performed the best out of all of the designs. Fig. 2c illustrates that two streams of green and red ink were simultaneously driven into the mixer, and that dark ink flowed out of the pre-treater within 10 s, indicating that the red and green inks had mixed together successfully. 3D mixers were also constructed using paper-cutting/folding, which appeared to be much more flexible than the 2D format (Fig. 2c). The separator and dilutor components were used to separate and converge the fluids in a certain ratio. Fig. 2d shows that green ink could be diluted into series concentrations. This component was useful for diluting original samples. Reactors were constructed to carry out biochemical reactions. A large surface-to-volume ratio and a high capacity for water retention are important for the performance of the reactor (Fig. 2e2). Detectors were constructed from a porous membrane and used to determine an unknown target in a sample. Various types of detection techniques could be combined with this system, including naked eye detection, electro-chemical detection and Raman spectroscopy, based on the real application.

Fig. 2 Demonstration of the various functional components (a) sample inlet created by cutting a spiral-like microchannel on thermoplastic sheet. (b) The sample pre-treater was fabricated as a rectangular piece of cellulose paper. (c) 2D mixers of different shapes, including (1) oval, (2) sausage and (3) circular. (d) (1) 3D mixers, (2) dilutors and (3) separators. (e) (1) Pumps were made by stacking circular paper, (2) reactors and (3) detectors.

The pumps in this system were used as a transporting force to accelerate the fluid circulating through the system (Fig. 2e1). A pump was constructed by stacking numerous pieces of circular cellulose paper, where the top-pad was the input port and the bottom-pad was the pump output port. The basic principle of this pump is based on fluid microgravity. When the paper was pressed, the fluid underneath it squeezed and pumped out of the bottom-pad. When the force was released, the fluid was absorbed and flowed from the top-pad. By applying this technique, the fluid could be continuously pumped via press and release cycling. Although manual operation was still required, other forces, such as electro-power or electro-magnetism, could also be adapted instead of manual operation. Apart from those mentioned, other relevant components could also be developed and integrated into this system, including continuous-flow mixers, continuous-flow micro-reactors, separators and electrophoresis components. These paper-based functional components could perform various types of bio-functions and analysis, which could be potentially applied to the analytical sciences.

System validation for the rapid detection of HIV

In this study, we developed a type of assay for the rapid detection of human immunodeficiency virus (HIV) based on this novel system, and integrated this with the gold nano-particle immunoassay (double antigen sandwich method). As shown in Fig. 3a, when the standard antibody of HIV (1 + 2) (provided in the commercial “diagnostic kit for antibodies to Human Immunodeficiency Virus (ELISA)”, Beijing Kinghawk Co. Ltd.) from inlet A and the functionalized gold nanoparticles (bio-functionalized with HIV gp41 antigen, Beijing Kinghawk Co. Ltd.) from inlet B meet together in the mixer micro-zone, bio-complexes of the HIV antibody (1 + 2)-gp41-gold nanoparticles were generated and transported into the following circular detection zone. This circular detection zone is composed of two layers (the upper layer was coated with the specific HIV antigen gp36 (Beijing Kinghawk Co. Ltd.), while the bottom layer was used to transport the fluid via capillary force). The HIV antibody (1 + 2)-gp41-gold nanoparticles-gp36 forms at the detection zone and a naked eye visible red dot could be detected when the positive HIV antibody exists in the sample, while a negative signal displays nothing in the detection zone.

Fig. 3 The novel micro-system for the rapid detection of HIV. (a) Schematics of the micro-system for the rapid detection of the HIV. (b) Detection limit (50 000, 5000, 500, 50, 5 and 0 ng mL⁻¹, respectively from left to right) and specificity (the standard antibody of HIV-1, HPV, HIV-2, HCV, HBV and positive serum, respectively from left to right) of the micro-system for the rapid detection of HIV.

We evaluated the detection limit and specificity of this novel assay in this study. As shown in Fig. 3b, the detection limit of this assay was 500 ng mL⁻¹ with naked eye detection during 5 min (which could be compared with the typical lateral flow technique) and could easily realize point-of-care detection, while the specificity mainly depended on the specific antigen (the antigen gp36 was available on the commercial market with high specificity) coated on the circular detection zone. Although this assay had a lower detection limit when compared with other sophisticated assays, the simple, flexible and low-cost characteristics made our assay much more attractive. In addition, when compared with the traditional methods of ELISA assay (Detection Kit for Human Immunodeficiency Virus Antibody, Beijing Kinghawk Co. Ltd.), the best advantages are the extensibility, flexibility and good consistency obtained in this study.

These tests demonstrated the potential usefulness of this paper-based novel micro-system in the analytical sciences.

To better describe the extensibility and real applications of this novel system, including much more various components, we perform a series of biochemistry functions for starch catabolism, including stage I, starch decomposition to maltose (eqn (1)), stage II, maltose decomposition to glucose (eqn (2)), and stage III, glucose oxidation (eqn (3)) (Fig. 4).

Fig. 4 Bio-microcircuit system for starch catabolism (a and a1) assembly of a bio-microcircuit for starch catabolism with red/green ink and a real starch sample, respectively. (b and b1) Replacement of the single detector of glucose with dilutors, with red/green ink and the actual starch sample, respectively.

This system of starch catabolism included sample/reaction buffer inlets, a pre-treater, a mixer, decomposition reactors, pumps, and dilutors, which were connected using the bio-microcircuit channels. In this functional bio-microcircuit system, the starch sample was introduced into sample inlet a1, whereas the reaction buffer was transported from inlet a2. These two fluids were mixed in mixer b and were then derived into the intestine-like reactor I to perform starch decomposition, followed by decomposition into the liver-like reactor II. Pump e was able to drive the fluid to circulate around the components c and d, and finally to circular reactor III for glucose oxidation and detection. Reactors I, II, and III were soaked in α-amylase, α-D-glucoside glucohydrolase, and glucose oxidase, respectively, for approximately 12 h. Reactor III was used to detect the catabolism of glucose. We could also replace the spherical reactors with dilutors for a semi-qualitative analysis of the decomposition product.

Apart from the application displayed, this micro-system was capable of integrating various other kinds of functional components in the circuit board in a flexible manner, which we think is much more attractive when compared to the planar and homogenized system of common microfluidics.^20–29 Most traditional microfluidic systems realize their functions via special planar and homogenized micro-channel structures. However, our proposed system, with the nature of a flexible 3D structure, is composed of two parts; one is the fluid circuit board, acting as the platform to realize the transportation and controlling of fluid, while another part is the functional components, used to perform all detailed bio-functions. We are looking forward to establishing a standard circuit board and various kinds of functional components with which we can design a functional micro-system by ourselves for special applications, such as bio-molecule purification, genetic analysis,³⁰ heavy metal detection,³¹ sperm cell analysis,³² biochemical reaction optimization, and so forth. We think that this novel micro-system could attract a lot of attention and lead to the development of microfluidics.

Conclusion

In this study, a novel micro-system has been introduced. This system, inspired by a microcircuit system with the nature of a flexible 3D structure, composed with a fluid circuit board and various functional components, was super powerful compared with typical planar and homogenized microfluidics. This novel system was validated through the rapid detection of human immunodeficiency virus (HIV) and starch catabolism. We believe that this novel system will open up new areas for applications in analytical science.

Acknowledgements

We are grateful for the kind help from colleagues in our groups. We acknowledge the Introduce Talents of Fudan University research funding (IDH1615001), the National Natural Science Foundation of China (21505024, 21427806, 21175029, 21335002, 81472032) and the Shanghai Leading Academic Discipline Project (B109), Shanghai Pujiang Program (14PJ1431400), Medicine Disciplines Leaders Project of Pudong District of Shanghai (PWRd2012-09) and Key Specialty Construction Project of Pudong Health and Family Planning Commission of Shanghai (Grant No. PWZz2013-03). In addition, we thank Prof. Xinyu Jiang for helping us review this article and polish the English language.

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