Patent Application
20050214799
This application claims the benefit of Korean Patent Application No. 2004-882, filed on Jan. 7, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a device and method for printing biomolecules onto a substrate using an electrohydrodynamic (EHD) effect, and more specifically to a device and method for printing biomolecules onto a substrate using an EHD effect by dropping a solution of biomolecules such as nucleic acids (such as probe DNA, RNA, peptide nucleic acid (PNA) and LNA), proteins (such as antigen and antibody), and oligopeptides, onto a solid substrate surface and fixing the biomolecules onto the substrate, to produce a biochip or a DNA microarray.
2. Description of the Related Art
As the human genome project makes great progress, there is an increasing need for methods for rapidly providing a large amount of genetic information for the diagnosis, treatment and prevention of genetic disorders. Since Sanger's method was used to analyze a sequence of nucleotides, a polymerase chain reaction (PCR) method for the reproduction of DNA has been developed and automated. However, the PCR method is troublesome and demands much time, labor, skill and expense, and thus can not be used to analyze a large number of genes. Thus, there has been a continuing search for new systems of analyzing a sequence of nucleotide, and for several years there has been advances in many fields relating to the manufacture and application of a biochip or DNA microarray.
A biochip or DNA microarray refers to a chip manufactured by microarraying oligonucleotide probes, each probe having a known sequence of up to hundreds of nucleotides, at hundreds to hundreds of thousands of predetermined positions on a solid surface made of, for example, silicon, surface-modified glass, polypropylene, or activated polyacrylamide. If a fragment of target DNA to be analyzed is applied to the biochip or DNA microarray, the target DNA complementarily hybridizes to the oligonucleotide probes immobilized on the biochip or DNA microarray. The hybridization is optically or radiochemically detected and analyzed to identify the nucleotide sequence of the target DNA, which is called sequencing by hybridization (SBH).
Use of the biochip or DNA microarray reduces the size of a DNA analysis system and enables genetic analysis with a trace of sample. In addition, multiple sequences of a target DNA can be simultaneously analyzed, thereby rapidly providing the genetic information of the target DNA at low cost. The biochip or DNA microarray can analyze a large amount of genetic information simultaneously within a short period of time and reveal correlation between the genes. Accordingly, the biochip or DNA microarray is expected to have many applications, for example, in genetic disorder and cancer diagnosis, mutant and pathogen detection, gene expression analysis, drug discovery, etc. In addition, the biochip or DNA microarray can be used as a microorganism or pollutant detector to find antidotal genes and also to produce antidotes on a large scale based on genetic recombination technologies. The biochip or DNA microarray can lead to great improvements in most biological industries, including the production of medicinal crops or low-fat meat.
Biochips or DNA microarrays can be either an oligo-chip or a CDNA chip, according to the type of probes immobilized thereon. Also, biochips or DNA microarrays can be manufactured by lithography, pin spotting, or ink-jet spotting.
However, the method using a pin has many problems. After biomolecules are printed with the pin, the pin is washed and then used to print other biomolecules. Thus, at least 99% of the biomolecules are wasted. Also, the size of the spots is somewhat irregular; sometimes the pin is blocked, and the life span of the pin is not long. Furthermore, the concentration of biomolecules changes depending on spotting time and it takes long time to print using capillary necking. First of all, there is a need for correct alignment in this method. Also, if the inkjet method uses a bubble jet process, it is necessary to use a momentary ultrahigh temperature. Thus, heat-sensitive biomolecules are likely to be adversely affected and the nozzle can become blocked.
To overcome the problems, a method for printing biomolecules onto a substrate using an electric field has been reported, for example, in EP 1157737. Using the fact that a probe DNA solution has a negative charge, this method involves applying a negative charge across a capillary holder 120 and a positive charge to a substrate 130 using a voltage applying unit 160, thereby dropping the probe DNA solution 103 from a capillary 110 onto the substrate 130 by attraction force.
However, the device and method for printing biomolecules onto a substrate using the electric field has the problem that the substrate must be always positively charged, and accordingly an alternating current (AC) voltage may not be used. In practice, the DNA solution comprises a various salts, thereby having a substantial positive charge. Thus, when only a direct current (DC) voltage is applied to the device, the probe DNA solution cannot be printed well, causing difficulty in practical use. Furthermore, proteins are not necessarily always negatively charged, and some nucleic acids, for example, peptide nucleic acids (PNAs) have neutral charges, and thus it is impossible to apply the device and the method to those biomolecules.
The present invention provides a device and method for printing biomolecules onto a substrate using an electrohydrodynamic (EHD) effect. The advantages of the device and method are that the process is quick, the biomolecule spots are easily aligned, and especially it is possible to spot the biomolecules correctly and reproducibly even when the printer body is not fully aligned with the substrate. In addition, the device and method are applicable to proteins which are sensitive to heat, and biomolecules which have neutral charges.
The present invention also provides a device and method for printing biomolecules onto a substrate using an EHD effect, permitting integration of silicon.
According to an aspect of the present invention, there is provided a device for printing biomolecules onto a substrate using an EHD effect, comprising; at least one capillary having an outlet through which a feeding solution of biomolecules selected from the group consisting of nucleic acids, proteins, and oligopeptides is discharged, the nucleic acids being selected from the group consisting of probe DNA, RNA, peptide nucleic acid (PNA), and LNA and the proteins being selected from the group consisting of antigen and antibody; a printer body supporting the at least one capillary; a substrate below the outlet having a target surface onto which the biomolecules are deposited; a first electric field forming electrode located on the printer body around the circumference of the outlet; a second electric field forming electrode spaced apart from the first electrode by a predetermined distance; and a voltage applying unit which is electrically connected to the first electrode and the second electrode to apply an alternating current (AC) voltage between the first electrode and the second electrode so that an electric field may be formed around the biomolecule solution suspended in the outlet, and due to the interaction of the electric field and a difference in dielectric constant between the biomolecule solution having a free surface and the surrounding atmosphere, the electric force acts inward on the biomolecule solution from the surroundings, thereby dropping a predetermined amount of the biomolecule solution onto the target surface of the substrate.
The voltage applying unit is capable of applying an AC voltage and a direct current (DC) voltage simultaneously, and the DC voltage can be applied substantially simultaneously when the AC voltage is applied for forming an electric field. This is advantageous for reducing the time of printing biomolecules.
In general, the DC voltage is in the range of 500 to 300,000 V and the AC voltage is in the range of 500 to 300,000 V.
The AC voltage may have a frequency of 40 to 1,000 Hz.
The substrate may be made of silicon. This is advantageous since it permits integration of silicon.
The first electric field forming electrode may be a circular electrode made of gold.
The device may further comprise a circular conductive band opposite the first electric field forming electrode and surrounding the target surface on the substrate.
In this case, the circular conductive band is approximately perpendicular to the outlet.
Also, the circular conductive band may be the second electric field forming electrode and may be made of gold.
The second electric field forming electrode may be located in a stage supporting the substrate.
The substrate surface inside and outside the circular conductive band may be hydrophobic-treated so that the contact angle of the solution with the surface is large enough to prevent the solution from flowing outwards.
A plurality of capillaries may be supported by the printer body, and the outlets of the capillaries may be arranged at the same pitch as a plurality of target surfaces on the substrate. This is advantageous since it is possible to print many and various types of biomolecules at the same time.
According to another aspect of the present invention, there is provided a method for printing biomolecules onto a substrate using an EHD effect, comprising the operations of; feeding a solution of biomolecules selected from the group consisting of nucleic acids, proteins, and oligopeptides to a capillary having an outlet through which the biomolecule solution is discharged, the nucleic acids being selected from the group consisting of probe DNA, RNA, PNA, and LNA and the proteins being selected from the group consisting of antigen and antibody; and applying an AC voltage between a first electric field forming electrode around the circumference of the outlet and a second electric field forming electrode spaced apart from the first electrode by a predetermined distance from a voltage applying unit capable of applying an AC voltage which is electrically connected to the first electrode and the second electrode, so that an electric field may be formed around the biomolecule solution suspended in the outlet, and due to the interaction of the electric field and a difference in dielectric constant between the biomolecule solution having a free surface and the surrounding atmosphere, the electric force acts inward on the biomolecule solution from the surroundings, thereby dropping a predetermined amount of the biomolecule solution onto a target surface of a substrate below the outlet.
In the operation of applying an AC voltage, the DC voltage may be applied substantially simultaneously with the AC voltage. This is advantageous for reducing the time of printing biomolecules.
In general, the DC voltage is in the range of 500 to 300,000 V and the AC voltage is in the range of 500 to 300,000 V.
The AC voltage may have a frequency of 40 to 1,000 Hz.
The substrate may be made of silicon. This is advantageous since it permits integration of silicon.
The first electric field forming electrode may be a circular electrode made of gold.
A circular conductive band may be further located opposite the first electric field forming electrode and surrounding the target surface on the substrate.
In this case, the circular conductive band is approximately perpendicular to the outlet.
Also, the circular conductive band may be the second electric field forming electrode and may be made of gold.
The second electric field forming electrode may be in the form of a plate located in a stage supporting the substrate.
The substrate surface inside and outside the circular conductive band is hydrophobic-treated so that the contact angle of the solution with the surface is large enough to prevent the solution from flowing outwards.
A plurality of capillaries may be supported by the printer body, and the outlets of the capillaries may be arranged at the same pitch as a plurality of target surfaces on the substrate. This is advantageous since it is possible to print many and various types of biomolecules at the same time.
The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:
The invention now will be described in detail with reference to the accompanying drawings.
The solution of biomolecules such as nucleic acids (such as probe DNA, RNA, peptide nucleic acid (PNA) and LNA), proteins (such as antigen and antibody), and oligopeptides is fed to the capillary 10 and stored therein. The capillary 10 is a tube with an outlet 11 at its bottom. The diameter of the tube is very small, allowing the surface tension of the biomolecule solution 3 to suspend it in the outlet 11.
The printer body 20 supporting the capillary 10 may be made of polymethlymethacrylate (PMMA). Whereas the printer body 20 supports one capillary 10 in this embodiment of the present invention, in general the printer body 20 supports a plurality of capillaries 10.
The substrate 30, comprising a biochip or DNA microarray, has a target surface 31 onto which the biomolecules are printed. The substrate 30 is made of silicon in this embodiment of the present invention, but of course, the silicon may be replaced with glass, indium tin oxide (ITO) coated glass, plastic, or other suitable materials. The surface of the substrate 30 inside and outside the circular conductive band 70 is hydrophobic-treated so that the contact angle of the biomolecule solution 3 with the surface is large enough to prevent the biomolecule solution 3 from flowing outwards.
The first electric field forming electrode 40 for forming an electric field is located on the printer body 20 around the circumference of the outlet 11, and is electrically connected to the voltage applying unit 60 via a first electrode lead wire 41. In this embodiment, a circular electrode made of gold is used as the first electric field forming electrode 40, but any other suitable conductor can be used, for example, platinum, copper, conductive polymers, or carbon nano tubes.
The second electric field forming electrode 50 is spaced apart from the first electrode 40 by a predetermined distance. If a voltage is applied, the second electrode 50 forms an electric field together with the first electrode 40. In this embodiment, the second electrode 50 is a circular conductive band 70 opposite the first electrode 40 and surrounding the target surface 31 on the substrate 30. The circular conductive band 70 must be approximately perpendicular to the outlet 11. The circular conductive band 70 is electrically connected to the voltage applying unit 60 via a second electrode lead wire 51. The circular conductive band 70 is made of gold in this embodiment, but any other suitable conductor can be used, for example, platinum, copper, conductive polymers, or carbon nano tubes.
The voltage applying unit 60 is electrically connected to the first electrode 40 and the second electrode 50 to apply an AC voltage between the two, forming an electric field around the biomolecule solution 3 suspending in the outlet 11. Due to the interaction of the electric field and a difference in dielectric constant between the biomolecule solution 3 having a free surface and the surrounding atmosphere, the electric force acts inward on the biomolecule solution 3 from the surroundings, thereby dropping a predetermined amount of the biomolecule solution 3 onto the target surface 31 of the substrate 30. The voltage applying unit 60 has a structure capable of applying an AC voltage and a DC voltage simultaneously at a specific frequency. In this embodiment, the DC voltage is applied substantially simultaneously with the AC voltage, thereby reducing the time to print biomolecules onto the substrate 30. The DC voltage is in the range of 500 to 300,000 V and the AC voltage is in the range of 500 to 300,000 V, and the AC voltage has a frequency of 40 to 1,000 Hz.
With regard to this constitution of the invention, referring to
In the first operation, the solution 3 of biomolecules such as nucleic acids (such as probe DNA, RNA, PNA and LNA), proteins (such as antigen and antibody), and oligopeptides is fed to the capillary 10 having the outlet 11 through which the biomolecule solution 3 is discharged. The capillary 10 is a tube with an outlet 11 at its bottom. The diameter of the tube is very small, allowing the surface tension of the biomolecule solution 3 to suspend it in the outlet 11.
Next, the AC voltage and DC voltage are simultaneously applied between the first electrode 40 and the second electrode 50 by the voltage applying unit 60, forming an electric field around the biomolecule solution 3 suspended in the outlet 11 as illustrated in
If the electric field is applied around the biomolecule solution 3 suspended in the outlet 11 by surface tension applied at a tangent to the circumference of the outlet 11, exceeding gravity, a curved electric potential line is distributed around the biomolecule solution 3 having a contact angle and a radius of curvature as illustrated in
Thereafter, the distribution of the electric field becomes more graduated around the droplet of the biomolecule solution 3, with the upper portion changing to form a groove as illustrated in
Hereinafter, embodiments of printing with the devices for printing biomolecules onto a substrate using an EHD effect according to the present invention will be described in detail with reference to
The printer body 20 was manufactured using polymethlymethacrylate (PMMA) and the circular conductive band 70 around the outlet 11 was made of copper, as illustrated
The experiments were performed with distilled water, a probe DNA solution, and a solution of protein, HBV antibody.
First, printing was performed with distilled water. Voltage from the voltage applying unit 60 was applied using the lower electrode as a ground electrode and the upper electrode as a working electrode. The voltage was set to 1000 V DC and 1000 V AC at 50 Hz and when a droplet was formed as illustrated in
Secondly, printing was performed with probe DNA. For this, perfect matched probe DNA oligomer (WP MODY3 EXON 3-6, C6NH2-5′-CGGAGGAACCGTTTC-3′) was prepared. 100 μM of the probe DNA, 100 μM of PEG, 3 μM of Cy5 activated ester were dissolved in a DMSO solution. Then, the solution was tested under the same conditions in the first experiment, and the same result was obtained.
Thirdly, the experiment was performed using HBV antibody dissolved in phosphate buffered saline (PBS) under the same conditions in the first experiment, and again, the same result was obtained.
The device for printing biomolecules onto a substrate using an EHD effect according to the embodiment as illustrated in
First, EHD printing was performed with distilled water. Voltage from the voltage applying unit 60 was applied using the lower electrode as a ground electrode and the upper electrode as a working electrode. The voltage was set to 1000 V DC and 2000V AC at 50 Hz and when a droplet was formed as illustrated in
Secondly, printing was performed with probe DNA. For this, perfect matched probe DNA oligomer (WP MODY3 EXON 3-6, C6NH2-5′-CGGAGGMCCGTTTC-3′) was prepared. 100 μM of the probe DNA, 100 μM of PEG, 31M of Cy5 activated ester were dissolved in a DMSO solution. Then, the solution was tested under the same conditions in the first experiment, and the same result was obtained.
Thirdly, the experiment was performed using HBV antibody dissolved in PBS under the same conditions in the first experiment, and again, the same result was obtained.
The device for printing biomolecules onto a substrate using an EHD effect according to the embodiment as illustrated in
First, EHD printing was performed with distilled water. Voltage from the voltage applying unit 60 was applied using the lower electrode as a ground electrode and the upper electrode as a working electrode. The voltage was set to 1000 V DC and 2500V AC at 50 Hz and when a droplet was formed as illustrated in
Secondly, the experiment was performed with probe DNA. For this, perfect matched probe DNA oligomer (WP MODY3 EXON 3-6, C6NH2-5′-CGGAGGMCCGTTTC-3′) was prepared. 100 μM of the probe DNA, 100 μM of PEG, 3 μM of Cy5 activated ester were dissolved in a DMSO solution. Then, the solution was tested under the same conditions in the first experiment, and the same result was obtained.
Thirdly, the experiment was performed using HBV antibody dissolved in PBS under the same conditions in the first experiment, and again, the same result was obtained.
As mentioned above, the device for printing biomolecules onto the substrate 30 using an electrohydrodynamic (EHD) effect comprises at least one capillary 10 having an outlet 11 through which the biomolecule solution 3 is discharged; the printer body 20 supporting the capillary 10; the substrate 30 below the outlet 11 having a target surface 31 onto which the biomolecules are deposited; the first electric field forming electrode 40 around the circumference of the outlet 11 on the printer body 20; the second electric field forming electrode 50 spaced apart from the first electrode 40 by a predetermined distance; and the voltage applying unit 60 electrically connected to the first electrode 40 and the second electrode 50 to apply an AC voltage between the first electrode and the second electrode to form an electric field around the biomolecule solution 3 suspended in the outlet 11, and due to the interaction of the electric field and a difference in dielectric constant between the biomolecule solution 3 having a free surface and the surrounding atmosphere, the electric force acts inward on the biomolecule solution 3 from the surroundings, thereby dropping the biomolecule solution 3 onto the target surface 31 of the substrate 30. The advantages of the device are that the process is quick, the biomolecule spots are uniform and easily aligned, and especially it is possible to spot the biomolecules correctly and reproducibly even when the printer body is not fully aligned with the substrate. In addition, the device is applicable to proteins which are sensitive to heat and biomolecules which have neutral charges.
In the Embodiments 1, 2 and 3, the simultaneous application of an AC voltage and a DC voltage between the first electric field forming electrode 40 and the second electric field forming electrode 50 to generate an electric field is described in detail. However, it is of course possible to apply only a DC voltage if required.
Although a circular electrode was used as the first electric field forming electrode 40 in the Embodiments 1, 2 and 3, various types of electrode can also be used.
As mentioned above, according to the present invention, using the device and method for printing biomolecules onto the substrate using an EHD effect, the process is quick, the biomolecule spots are uniform and easily aligned, and especially it is possible to spot the biomolecules correctly and reproducibly even when the printer body is not fully aligned with the substrate. In addition, the device and method are applicable to proteins which are sensitive to heat and biomolecules which have neutral charges.
Further, the present invention uses a substrate made of silicon, thereby permitting integration of silicon.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-882 | Jan 2004 | KR | national |
