Patent Grant
9242244
Pipette tip columns contain functionalized solid material in a column formed at the end or lower part of the tips. The columns are used to separate and purify sample materials from a variety of sources including biological samples and environmental samples. Pipette tip columns are often used with robotic liquid handlers. However, robotic liquid handlers can cost up to several hundred thousand dollars which is a very large of investment for many users. Therefore, there is a need for a simplified, lower cost, lower throughput means for reliable operation of pipette tip columns.
An apparatus and method of using a freestanding pipette with pipette tip columns were developed. The pipette tip columns are used for performing separations such as solid phase extraction. The pipette is operated with the pipette tip columns inserted into the wells of a multiwell microplate. In this configuration the pipette is freestanding and will not tip over. The open lower ends of the pipette tip columns are approximately centered within the plate well. The columns and plate are designed in such a way that the open lower ends of the pipette tip column are in contacts with liquid in the plate well however, the columns do not seal on the well bottom, preventing flow in and out of the column. The pipette contains the appropriate firmware and software to control flow for all steps of pipette tip column operation.
The present invention provides a device and method for performing separations with a pipette tip column. The device is a hand-held freestanding pipette that can operate a plurality of columns simultaneously in combination with pipette tip columns and a microplate.
An advantage of the pipette of the invention is that it can perform parallel operation of pipette tip columns yet it is significantly less expensive than a robotic liquid handling system. Another benefit of the device is that it is similar in size to a multichannel pipette and therefore does not occupy much laboratory bench space. An additional advantage of the pipette of the invention is that it is freestanding. That is, a stand is not required for its operation.
Although it is desirable to operate pipette tip columns with a handheld electronic pipette, existing electronic pipettes have limited keyboards and displays and limited software, firmware, memory and micro-processing capabilities. PhyNexus, Inc. (San Jose, Calif.) sells the ME200 and ME1000 Purification Systems for semi-automated processing of 1-12 samples at a time. These systems are comprised of a pipette held in place on a stand and controlled via Windows-based software. The ME system allows automated programming of an 8 or 12 channel pipette with complete purification of up to 12 samples in as little as 15 minutes.
The ME Purification System is quite useful however, it has some drawbacks. Although the ME pipette stand system is much lower cost than robotic liquid handlers, the investment is still several thousand dollars. It can be difficult to adjust the ME and it can be complicated to use. The ME pipette technology is based on a computer controlled pipette that is placed in a stand and connected to the computer through a cable. The computer was needed because the software was too complex and lengthy for loading onto an electronic pipette. However, the presence of the cables can be cumbersome, and a self-contained device is preferable. Furthermore, the ME requires manual adjustment of the z-position which can be time-consuming and runs the risk of being inaccurate.
Therefore, there exists a need for a device (and accompanying method) in which the lower end of the pipette tip column(s) is centered in a microplate well or tube at the proper height for pipetting small volumes of liquid. This device should hold the pipette tip column at the appropriate height to prevent sealing the lower end of the column against the well bottom. Additionally, the device should not require manual adjustment.
To overcome the drawbacks of existing systems, an apparatus and method of using a pipette with pipette tip columns were developed. The apparatus is a free-standing or standless pipette with pipette tip column(s) containing firmware, software and firmware control capable of going through all the steps of purification with pipette tip columns and a deep-well plate. The columns and plate are designed to match so that the pipette with pipette tip columns attached stand vertically when placed in the plate and does not tip over. The columns and plate are designed so that the ends of the pipette tip columns are substantially self centering but do not seal on the plate bottom.
Several factors had to be developed, solved, tested, verified and determined to actually operate in order to be able to use the standless electronic pipette, pipette tip column and microplate of the invention. It is counterintuitive to operate a pipette without holding it. In fact, electronic pipettes are also called handheld pipettes; their name describes what they are, how they are designed and how they are used. Obviously, if a pipette holding pipette tip columns is not supported, the pipette and columns will fall over. In addition, pipette tip columns usually require several steps of operation with different solutions which requires moving the pipette to a series of vials or wells. These steps are traditionally done with the firm support of a hand.
A series of experiments was performed in an attempt to balance the pipette and pipette tip columns with minimum support. It was found that the most favorable balancing of the pipette could be achieved by keeping the pipette as close to vertical as possible. If the pipette was positioned at an angle, then the off-center weight of the pipette would simply pull the whole apparatus over.
The second problem was maintaining the pipette with pipette tip columns in a (more or less) vertical position without a stand or support. The initial solution to this problem was the use of deep-well plates designed to fit the size of the columns. However, pipetting operations are not usually performed by simply placing a pipette into a deep-well plate. The bottom of the pipette tip could seal and prevent flow. Coating the outside of the wall of the pipette tip with liquid could increase the volume of solution aspirated or could contaminate the solution. The same problem could be expected when pipette tip columns were substituted for pipette tips.
A third potential problem was the weight of the pipette pushing the lower end of the pipette tip columns too far down into the well, sealing the end of the columns and preventing flow in and out of the column. In hand-held pipetting operations, the pipette tip can be held at an angle to prevent sealing of the bottom of the tip. In a robotic system, the tips come straight down but the depth or z-axis position is controlled by computer so that the ends of the tips do not come down too far, sealing the ends of the columns.
The size of the plate, the diameter of the wells, shape of the wells relative to the diameter of the pipette columns were chosen to keep the pipette and pipette tip columns more or less vertical and stable from falling when placed into the deep-well plate. It was found that increasing the depth of the wells in 96-well deep-well plates could keep the columns more or less vertical. In certain embodiments, the deep well plates are in the range of 20 mm to 45 mm. In some embodiments the height of the plate is at least 22 mm, at least 27 mm, at lease 31 mm, at least 41 mm, at least 42 mm, at least 43 mm or at least 44 mm.
The diameter of the column relative to the opening also had to be considered although as the depth of the well was increased the diameter of the well relative to the column became less important. The diameter of the columns could not be too small relative to the diameter of the wells in the plate. Inserting the pipette with column or columns into the deep-well plate kept the pipette from tipping by keeping it standing more or less vertical. If the pipette is at an angle more than 25-45 degrees from vertical, it would likely not be stable. In preferred embodiments, the angle of the pipette is 35 degrees or less from vertical (perpendicular to the plate). For example, the angle of the pipette can be less than 35 degrees, less than 30 degrees, less than 25 degrees, less than 20 degrees, less than 15 degrees, less than 10 degrees, less than 5 degrees, less than 4 degrees, less than 3 degrees, less than 2 degrees or less than 1 degree from vertical.
In preferred embodiments, the plate is a 96-well deep-well microplate in ANSI or SBS format. In other embodiments, a non-standard plate format or even a custom plate could be used. In certain embodiments, the plate could have fewer than 96 wells. In those embodiments, the plate could be comprised of 6, 12, 24, 48, 192 or 1536 wells.
In certain embodiments, the microplates used have quite shallow wells and are not considered deep-well plates. In these embodiments, the plate height can be less than 22 mm, less than 20 mm, less than 10 mm or even less than 5 mm. In still another embodiment, tubes or vials can be substituted for a microplate.
A fourth problem to be solved was programming the pipette specifically for operation of pipette tip columns. Pumping solutions through pipette tip columns is quite different from simply aspirating and expelling liquids. The presence of the solid phase in the tip can give the column back pressure. In preferred firmware and software embodiments, time pauses are programmed at the end of some aspiration and expel pumping strokes. This is preferred if there is appreciable column backpressure and the flow through the column is slowed or delayed from the pumping stroke.
Sometimes, engagement of the pipette tip column with the pipette can create a positive pressure. This is particularly true when the column has high backpressure, for example, if the solid phase is wet such as is the case when using a hydrated gel resin and air cannot pass through the bed. If a positive pressure is present, programming may be used to compensate for this initial buildup of pressure. Pressure buildup on insertion of the column onto the pipette and column backpressure can increase as the column diameter decreases.
Expulsion of extra volumes at the end of each capture cycle and each wash cycle may be useful to ensure all of the liquid on top of the column bed is expelled before the column is moved to the next solution. But care must be taken as it is preferred that no air enter the bed of the pipette column even if extra pump volumes are used. Often, slower flow rates are used when pumping solutions through pipette tip columns than when simply aspirating and expelling liquids in an empty pipette tip.
Electronic pipettes often include a blow out at the end of the expulsion stroke to ensure that the liquid inside the tip is expelled. This operation is often included in the firmware and software and cannot be modified by the user. But the blow out may not be compatible with pipette tip column operation. The intake of liquid in the next stroke may be hindered by the introduction of air into the column bed by the blow out. The blow out may prevent or partially disrupt the aspiration of the liquid into the pipette tip column.
Most often, pipette tip columns are operated with back and forth flow. That is, liquids are aspirated and expelled only through the open lower end of the column. However, in certain embodiments, liquids can enter the column at the upper end and exit through the lower end, flowing in a single direction. In these embodiments, liquid may be added to the top of the pipette tip column and the pipette may be engaged to push liquid through the column. The pipette tip columns may be used for extraction and chromatography and may employ a number of different column chemistries.
In certain embodiments, the pipette tip columns may be used in a several step process. After an optional conditioning of the column, the column may be placed into a sample. One or more analytes from the sample can be captured by the solid phase within column with back and forth flow. Several capture buffer solution conditions and/or several column types may be surveyed by operating the columns in parallel.
After capture and expulsion of the sample liquid, the column can be placed into a wash solution to remove impurities. In some embodiments several different washes may be used to remove different types of bound or entrained impurities. Again, the effectiveness of different wash buffers may be surveyed by operating the pipette tip columns in parallel. In certain embodiments, the wash solution may be removed from the column with a water or saline solution to facilitate introduction of an acid elution solution.
The final step of extraction is elution of the purified analyte. The elution may be performed with serial increases with elution solvent strength to determine the optimum eluting solvent. In this embodiment, conditions may be identified that elute the compound of interest while retaining impurities. Several elutions can be performed to ensure the complete removal of the purified analyte.
All of these operations result in requirements of an electronic pipette that are quite different from simply aspirating and expelling liquids.
Furthermore, the standless pipette need not be limited to having the dimensions of those that are commercially available. The geometry of the standless pipette can be changed to suit the invention.
In some cases, the diameter of the pipette tip column is considerably smaller than an unmodified plate. In some cases, this will cause the pipette tip column and pipette to tilt from vertical causing the combination of pipette, pipette tip column and plate to tip.
An added benefit of using the adapter is that it can center the column in the well of the plate and in some cases, keep the column end from sealing at the plate well bottom by preventing the lower end column from settling completely into the plate. With a precise and accurate fitting of the column diameter with the diameter of the plate hole (width 3), the end of the column can be positioned to just above the bottom of the plate well, thus preventing the end of the column from being sealed at the well bottom.
Two or more adapters may be used for a multichannel pipette. It may not be necessary to employ an adapter in each well as long as two or more adapters are placed far enough apart to position all columns attached to the multichannel pipette similarly. The single channel adapter can also be used with a tube or vial. The tube or vial can be placed in a rack or other holding apparatus.
Although
It was discovered that supporting the plate or having a larger base support at the bottom of the plate improved stability also improved stability. Adding or securing a base to the 96 well plate increased surface area of the plate and the pipette with pipette tip columns was less likely to tip over. Increasing the area of the plate by at least 50%, 100% 200% up to 500% increased the stability of the pipette and pipette tip columns. However this was not enough to provide a secure system that did not tip over.
An adaptor or modifier can be used on top of the microplate to adjust the diameter of the wells. In some cases, the diameter of the pipette tip columns is small relative to the wells of the deep-well plate. In some embodiments, a plate adaptor or modifier can be placed on the deep-well plate or the pipette tip column that effectively narrows the diameter of the wells within the deep well plate. The attachment may also center the column in the well. This narrowing of the well diameter prevents the bottom of the pipette tip column from reaching and sealing at the bottom of the deep well plate. The attachment can be on 1 well, several wells, or all 96 wells.
One embodiment of the attachment is shown in
Use of the adaptor is not limited to deep-well plates. In some embodiments the microplate can be quite shallow, for example having a height of less than 2.2 cm, less than 2 cm, less than 1.5 cm, less than 1 cm or even less than 0.5 cm. The function of adaptor is to keep the standless pipette that is engaged with at least one pipette tip column, substantially vertical in the microplate, tube or vial.
Once balance and stability is achieved, it does not matter if one column or several columns are balanced. If more than one column is being balanced, but not all of the channels of the pipette are used, more secure balancing can be achieved by spreading the columns out across the multi-channel pipette. The system of pipette and pipette tip column can support 1 column, 1-8 columns 1-12 columns or 1-24 columns with the appropriate pipette. The pipette can be single-channel or multi-channel pipette.
Another technical problem was that it is very important to have the lower end of the pipette tip column very near the bottom of the well in the vial or plate without sealing the open lower end of the column. Otherwise, the ability to pick up of small volumes of liquid an pump them into the column would be inconsistent or impossible. The stand and liquid robotic handlers are designed and programmed to keep the tip of the column from touching the bottom and sealing. In fact, it is very easy to seal the bottom of the column and care must be taken not to do so.
The problem of sealing can be solved by carefully selecting the deep-well plate geometry to accommodate the column in the well. One solution is to select the shape of the well bottom so that a seal could not readily be formed. A diamond-shaped well bottom was used so that the round column tip could not seal on the well bottom. This configuration was found to allow the pickup of small drops of liquid. In fact, any irregular shape at the well bottom can be used to prevent sealing of the lower end of column, as long as the shape does not prevent complete aspiration of small liquid volumes.
The distance between the lower end of the pipette tip column and the well bottom can be particularly crucial when pipetting small volumes. The lower end of the pipette tip column can even be touching the well bottom as long as a seal is not formed. If larger volumes are aspirated and expelled, the distance between the lower end of the pipette tip column and the well bottom can be greater.
Another solution to the sealing problem is to select the combination of microplate and column in such a way that the column is positioned at the appropriate height. This can be accomplished by selecting the diameter of the column so that a friction fit or restriction of the column prevents the column from sealing on the bottom. However, the danger is that a seal could possibly be formed around the sides of the column in the deep-well chamber. Sealing of the chamber could cause development of a pressure (during the expel step) or vacuum (during the aspirate step) and disrupt fluid flow in and out of the column. This design had to be examined to determine if a detrimental seal around the column would be formed.
Another potential problem is that it could be difficult to remove the pipette tip columns from the plate if a seal were formed. So all of these potential problems were to be tested to make certain the ends of the columns did not seal while still maintaining the pipette in a position that was 45 degrees or less from perpendicular. In some embodiments, the pipette is 35 degrees or less from perpendicular.
It was also necessary to confirm that the working standless electronic pipette system with pipette tip column would produce a useable, pure extraction product. Pipette tips are not usually completely immersed in the liquids being transferred. In addition to the sealing issue, contamination could result from liquids covering the outside of the tip. It was unknown whether this issue would negatively impact the purity of the extracted analyte. The results of the testing after the complete apparatus was built, described in Example 1, show that it is possible to effectively purify protein with the columns immersed in sample and wash solutions.
In certain embodiments, the deep well plate can be secured to the work surface or to a base. In these embodiments, it is less critical that the pipette be completely vertical i.e. perpendicular to the deep well plate. Instead, the pipette can be in the range of between 1 degree and 45 degrees from perpendicular (vertical). Because the plate is secured, the pipette with pipette tip columns will not fall over. An advantage of positioning the pipette and columns at an angle is that the columns would not seal as easily against the bottom of the plate.
Any means can be used to secure the plate. When the plate is secured to a base, the base can be made of any “hard” materials including plastic, metal or a combination. The base should have sufficient area to keep the microplate from falling over when a pipette and tip(s) are inserted into the plate. The base can accommodate one or more microplates.
An embodiment of such a base is shown in
Another method of securing the plate is to have sliding pieces that move into place to hold the deep well block down. This embodiment can accommodate either SBS or ANSI format plates. For example, the microplate can be placed in the center of a base and plastic or metal pieces on runners or slides can slide toward the block and secure it with a friction fit. A third embodiment would be to have clamps on multiple sides that swivel toward the deep well block to provide a friction fit. This embodiment can be used with SBS or ANSI plate formats.
Pipette Firmware and Firmware Control
Electronic pipettes have a self-contained firmware that allows programming of the pipette to perform pipetting and mixing operations. The firmware includes the programs and data structures that internally control the pipette. Because of space and memory limitations, the programming is directed to the operations for which a pipette is intended e.g. pipetting (aspirating, expelling), transferring and mixing liquids.
The use of a pipette as a pump for pipette tip columns involves operations far more involved, complex and different from pipetting. This operations include slow control of the flow rate, pumping delays, control of the number of back and forth flow cycles, pump displacement volume, control of the blow out function e.g. not have a blow out or have a controlled blow out between capture and wash and between wash and elute, be able to change the plunger aspiration volumes for each step of extraction, capture, wash and elute, be able to add additional captures, washes, and elutions, and other functions if necessary. (Pipette blow out is the pipetting function where during expulsion, the piston of the pipette travels past the zero position pushing the last bit of liquid out of the pipette tip.) The pipette should also be able to direct or signal the user the step in the extraction process because the pipette must be moved manually from well to well containing the various capture, wash and elution liquids.
This operational control is not available or programmable on commercial pipettes. The invention of a freestanding electronic pipette required redesigning the firmware and the procedure used to program the pipette for use. The hand-held electronic pipette software is not compatible with the pipette tip column operation and at the outset, it was not known whether an electronic pipette could be redesigned. The following technical challenges were addressed and solved in the instant invention.
Examples of the number and types of steps are outlined in the Examples that follow. The steps and operations are much more complex than normal pipetting operations. In some cases, the plunger movement must be greater than the amount of liquid picked up and moved back and forth through the column. The programming must accommodate this when necessary. Firmware may have to be modified to prevent a blow out at the end of the expel cycle (except at the final expel for elution.) It would not be obvious to use an off the shelf electronic pipette because it would not work for pipette tip columns. Nor would it be obvious that a pipette with limited electronic capability could be modified as a free-standing apparatus used with columns and a deep-well plate.
The details the firmware design used to meet the goals of operating a pipette tip column are given in the various examples herein. For some types of columns, it is necessary to program extra aspiration and expulsion volumes. For some types of high back pressure columns, a delay at the end of each half cycle may be needed. If the back pressure of the column is low enough, then the delay at the end of each half cycle may not be needed. The flow rates can be less than what is used in normal pipetting operations. In some cases, the flow rates are up to 50 times slower than what is used in normal pipetting operations.
Types of Applications and Columns
A pipette tip column is defined herein as any column adapted to engage the barrel of a pipette either directly or indirectly. The invention can be used with any type of pipette tip column that uses pipette pressure to force liquid in and out of the column bed from the bottom of the column. The pipette tip column body can be a commercially-available pipette tip, a modified tip or it can be a custom column body. Any volume of pipette tip can be used. For example the pipette tip volume can be 1 μl, 5 μl, 10 μl, 20 μl, 50 μl, 100 μl, 200 μl, 500 μl, 1000 μl, 5 μl, 10 μl, 20 μl, 25 μl or more.
Examples of pipette tip column contents are a packed resin bed, disk, precipitated bed, monolith, media encapsulated in a fiber or polymer or a fluidized bed. Column resins include affinity resins, reverse phase, normal phase, hydrophobic interaction phase, ion exchange, silica, polymer, inorganic phases and others.
This apparatus and method of use can be used for different extraction methods including but not limited to Protein A, Protein G, Protein L or other antibody extractions, IMAC and similar resins for recombinant tagged molecules, antiFlag, Streptavidin, Avidin, reverse phase and ion pairing, reverse phase silica and polymeric normal phase, ion exchange and any resin that can be used in an extraction mode to extract nucleic acids, proteins, polypeptides, drugs, organic molecules, and inorganic molecules and other materials and molecules.
Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless so specified.
An experiment was performed comparing the technology of the invention with pipette tip columns used in a spin column mode and in a manual mode using a manual pipette. Pipette tip columns (PhyNexus, Inc.) were used in three different modes: (a) spin column/centrifuge, (b) manual pipette, and (c) standless electronic pipette. In each mode, an identical volume of the initial sample protein was purified by single-lots of IMAC, Protein G and Protein A pipette tip columns using identical wash and elution buffers. Protein samples consisted of either mouse IgG1, human IgG, His6-ubiquitin or His6-rubredoxin protein standards spiked into appropriate binding buffer processed using appropriate pipette tip columns containing Protein G, Protein A, or Ni-IMAC resin. Sample flow-through, wash flow-through and elution fractions were assessed for capture efficiency, purity and overall yield by quantitative HPLC analysis.
The pipette tip column used as a spin column in a centrifuge: Interaction between the sample and affinity resin in a pipette tip column when operated in spin column mode is limited to a single pass through the bed during the centrifugation step. Results obtained in this configuration exhibit the versatility of the pipette tip column format while at the same time demonstrating inherent limitations of the spin column process resulting from reduced contact between sample and resin. The purification efficiency for two His6-tagged proteins using Ni-IMAC pipette tip columns was measured and the purification efficiency of mouse IgG1 processed with Protein G and Protein A pipette tip columns was measured in all three methods.
The pipette tip column used in a hand-held manual pipette: Using a pipette to control the purification process allows increased contact with the resin by back and forth flow of the sample through the column. A manual pipette used with an identical column with same sample gave better capture and recovery of protein in a smaller volume compared to the spin mode. In general, results were as much as 65% better than those obtained when the pipette tip column was used as a spin column. Capture efficiency for two separate His6-tagged proteins using Ni-IMAC columns demonstrating reduced capture efficiency when samples are processed in the spin column mode. Capture efficiency is improved by increasing the number of capture cycles when processing pipette tip columns using a manual pipette. 4-6 cycles are normally adequate to capture the protein to equilibrium. However, it is very tedious holding the column and tip in the correct position throughout the pumping operations. If the column is held too high, some of the fluid in the vial or plate may not be pumped into the column. If the column is held too low, the end of the column may seal on the plate or vial and liquid may be prevented from flowing in or out of the column. The flow rate is also difficult to control using the manual pipette.
Pipette tip column used in a standless electronic pipette: An electronic pipette is used in a similar manner to the manual pipette but is free-standing with the columns contained in a deep-well plate. The electronic pipette firmware was modified so that it could be programmed to use precisely controlled back and forth flow rates, number of cycles, pause between capture and wash and between wash and elute while optionally adding more capture, wash and/or elution steps if needed. The piston position and aspiration and expulsion volumes were controlled relative to the volume of liquid passed through the column with controlled blow out at the end of the various operations. The standless electronic pipette used with pipette tip column gave superior recovery and purity over the other two methods tested. The results were on average, 130% better than spin columns at sample capture and 70% better than sample capture using manual operation.
The capture efficiency of mouse IgG1 on 80 μL Protein G columns was determined keeping all conditions the same and comparing the percent captured with spin column, manual pipette and standless electronic pipette (Table 1).
Table 2 shows results of the purification of two His-tagged proteins on IMAC resin keeping all conditions constant and comparing spin column and standless electronic pipette. The 500 μL samples consisted of either 0.9 μg His-rubredoxin in PBS buffer containing 0.05% Tween 20 or 5 μg His-ubiquitin in PBS buffer containing 0.05% Tween 20. The samples were processed by columns containing 80 μL of IMAC resin.
Columns were equilibrated with 500 μL PBS buffer. 500 μL samples consisting of 5 μg mouse IgG1 in PBS buffer containing 0.05% Tween 20 was captured by one of three methods. The spin method was carried out by adding the sample to the top of the columns and inserting the column into a 15 mL conical tube. This sample was forced through the column by spinning in a clinical centrifuge at ˜5K rpm for 30 seconds. For manual operation, the pipette was set to 480 μL and the plunger was depressed. The column was attached to the pipette while keeping the plunger depressed. The columns were submerged into the 500 μL sample keeping the pipette and column completely upright and the end of the column at the bottom of the sample. The plunger was released at the slow rate of 5 seconds to aspirate the full 480 μL. After aspiration, the pipette and column were held in the same position for 15 seconds. The plunger was next depressed at a rate of 5 seconds to completely dispense 480 μL. The pipette and column were held at the same position for 15 seconds. This consists of 1 cycle and the procedure was repeated for a second cycle. For standless Electronic Pipette operation, the manual method was repeated using the programming on the electronic pipette.
Table 3 shows the comparison of 5 μL and 80 μL bed volume columns of Protein A resin capturing and recovering human IgG with a manual pipette comparing 1, 2, 3, and 4 capture cycles. The sample consists of 200 or 500 μL for the 5- and 80-μL bed volume columns, respectively. Samples consist of 0.02 mg/mL human IgG (Sigma, I4506) in PBS buffer supplemented with 0.05% Tween 20. Aliquots were removed after each cycle and quantified by HPLC.
Table 4 compares the elution efficiency of mouse IgG1 from a 80 μL protein G resin column keeping everything the same with a spin column, manual pipette and standless electronic pipette. Columns were loaded as per Table 1 and washed with 500 μL PBS buffer followed by a second wash of 500 μL 140 mM NaCl. Columns were subjected to two elutions of 250 μL elution buffer, each, consisting of 200 mM sodium phosphate pH 2.5, 140 mM NaCl. Elutions were analyzed by quantitative HPLC.
Table 5 compares the elution of proteins from a 5 uL IMAC column using a spin column and the standless electronic pipette. 200 μL samples consisted of either 0.012 mg/mL His-rubredoxin or 0.012 mg/mL His-ubiquitin. Samples were captured as described and washed twice with 200 μL 5 mM imidazole in PBS buffer followed by two elutions of 15 μL of buffer containing 500 mM EDTA and 500 mM NaCl. Elutions were analyzed by quantitative HPLC.
In summary, although all three modes gave good recovery of a variety of proteins purified with Protein G, Protein A and Ni-IMAC affinity resins, in every case the pipette tip columns when used in the back and forth flow mode delivered superior results to the spin column mode. When used in the 96-well plate an important advantage to using pipette tip columns is the ability to contain and track samples and buffers systematically. The protocol is efficient and significantly less prone to errors. Finally, protocols using plates and pipette tip columns with manual or standless electronic pipettes enabled true parallel processing of multiple test samples alongside one or more controls. Such protocols minimize or even eliminate errors through simplified workflow and structured analysis of experimental results.
The volumes stated in this process are for guideline purposes only and can change depending on the volume of the sample, the size of the column, the extent and type of washing and the type and amount of elution volume. The descriptions apply the control needed for pipette tip columns by an electron pipette with the appropriate firmware, software and programming. The programming adjustments will apply to many different types of columns including packed bed, encapsulated bed and monolith columns and including gel resins, polymer resins and silica or other inorganic based resins. But in general the processing steps are optional conditioning, capture, washing, optional additional washing steps and enrichment or elution. All of these steps are normally programmed using a computer. In order to program these into an electronic free standing pipette, the pipette must be modified to contain the appropriate microprocessing ability, firmware programming and storage, software programming and storage and interface. The microprocessing power needed goes far beyond what is required for pipetting and mixing operations and must be designed into the pipette.
Condition Tip
This step is to ensure that the tip is in a uniform ready condition. This may involve treating with a solvent and/or removing excess liquid from the bed. This may be done at the factory or directly prior to using the column. If agarose or similar materials are used, the bed must be kept fully hydrated before use. Air may be introduced into the bed at this stage (or any stage). But because of the need to control the movement of the liquid through the bed, it is generally not preferred except at this stage.
Step 1. A particular volume of air is drawn into the syringe. The volume amount depends on the type of tip used (e.g., 1000+ tip or 200+ tip).
Step 2. The tip itself is attached to the system (e.g., handheld, ME 100)
Step 3. The same volume of air as in Step 1 is expelled.
Step 4. A particular volume of air is drawn into the syringe again. This extra volume is used in various later steps throughout the method to allow extra expulsion of liquid. Optionally the tip may be removed and reattached to equalize pressure within the column.
Capture (Sample Loading)
This step can be performed with bi directional flow and as many cycles as needed may be used to ensure maximum or desired uptake. High linear velocities are used to reduce time needed for loading. Because of this, it is likely that most of the loading interactions are at the surface of the packing material. The linear velocity may have to be lowered for slow extraction reactions. After the loading, the excess liquid is expelled.
Step 5. The handheld is lowered into vials filled with sample (e.g., 200 uL of sample for 200+ pipette tip column).
Step 6. A particular volume of sample is drawn into the syringe.
Step 7. The same volume is expelled (one cycle completed).
Step 8. The same volume is drawn again into the syringe.
Step 9. A volume slightly greater than Step 8 is expelled (two cycles completed).
Purification (Washing)
The wash cycle is used to remove excess matrix material or to remove lightly adsorbed or non specific adsorbed materials so that they do not come off in the elution cycle and contaminate the analyte material. The wash cycle can involve solvents or solvent having a specific pH or containing components that that help remove materials which interact lightly with the extraction phase. In some cases, several wash solvents might be used in succession to remove specific material. These cycles may be repeated as many times as necessary. In other cases, where light contamination can be tolerated, a wash cycle may not be used. If a wash step is used, one or more solvents may be used. This example shows two solvents.
PBS Wash
Step 10. The handheld is raised, and vials are replaced with fresh vials of PBS wash solution.
Step 11. The handheld is lowered to begin the wash mode.
Step 12. A particular volume of PBS wash solution is drawn into the syringe.
Step 13. The same volume of solution is expelled (one cycle completed).
Step 14. The same volume of solution is drawn into the syringe again.
Step 15. A volume slightly greater than Step 14 is expelled (two cycles completed).
Water Wash
Step 16. The handheld pipette is raised, and vials are replaced with fresh vials of water. The handheld is lowered to finish the wash mode.
Step 17. A particular volume of water is drawn into the syringe.
Step 18. A volume slightly less than Step 17 is expelled (one cycle completed).
Step 19. The same volume of water as Step 17 is drawn into the syringe.
Step 20. A volume slightly greater than Step 19 is expelled (two cycles completed). The tip may be removed and reattached to equalize pressure within the column.
Enrichment (Elution)
Elution or desorption of the analyte is performed with as small volume as possible to maintain the concentration of the analyte in the final solution. This cycle may be repeated as many times as necessary. Step elutions may be performed to remove materials of interest in a sequential manner.
Step 21. The handheld is raised, and vials are replaced with fresh vials filled with Elution Solution.
Step 22. The handheld is lowered to begin Elution Mode.
Step 23. A particular volume of elution solution is drawn into the syringe.
Step 24. The same volume of solution is expelled (one cycle completed).
Step 25. The same volume of solution is drawn again.
Step 26. The same volume of solution is expelled (two cycles completed).
Step 27. The same volume of solution is drawn again.
Step 28. The same volume of solution is expelled (three cycles completed).
Step 29. The same volume of solution is drawn again.
Step 30. A volume slightly greater than Step 29 is expelled (four cycles completed).
Step 31. Sample vials now contain purified and enriched protein.
This example is intended to illustrate how the firmware of an electronic pipette would be programmed to operate without computer control. The instructions could be used with Rainin handheld electronic pipettes such as a) EDP-3, SE-200, E8-200 and E-12-200 or b) EDP-3, SE-1000, E8-1000 and E-12-1000 for operation of pipette tip columns if this capability could be designed into these electronic pipettes. Appropriate terms and nomenclature would be different for different electronic pipettes or electronic pipette specifically designed for the use of pipette tip columns. The terms used in this example are chosen from those available with the display of these particular electronic pipette models.
Start operation, Set up deep-well plate with appropriate number and volumes of condition, sample, wash and elution volumes and aliquots. Program the pipette.
1. Hold down MODE until display flashes, Scroll with MODE until “PHY OFF” is displayed. Use ARROWS to select “ON.” Press RESET to activate PhyNexus operation mode.
2. To run the saved program, go to step 12, or begin reprogramming at Step 3.
3. Press RESET to display “FLO.” Use ARROWS to set Flow Rate of 1=Low, 2=Medium or 3=High. Medium speed is recommended.
4a. Press RESET to display “CAP.” Use ARROWS to set the Capture Volume between 40-200 uL.
4b. Press RESET to display “CAP.” Use ARROWS to set the Capture Volume between 230-1000 uL
5. Press RESET to display CAP nbr1. Use ARROWS to set the number for capture fractions 1-2, equivalent to the number of wells containing sample aliquots.
6. Press RESET to display CAP CYC1.” Use ARROWS to set the number of Capture Cycles per well to 1-8. Each sample well will be processed by this number of cycles. 4 capture cycles are recommended.
7a. Press RESET to display “Pur.” Use ARROWS to set Wash Volume to 40-200 uL.
7b. Press RESET to display “Pur.” Use ARROWS to set Wash Volume to 230-1000 uL.
8. Press RESET to display “Pur nbr1.” Use ARROWS to set Number of Washes to 1 or 2, equivalent to the number of separate wash wells. Each wash well will be processes by 2 wash cycles.
9a. Press RESET to display “ELU.” Use ARROWS to set Elution Volume to 10-200 uL.
9b. Press RESET to display “ELU.” Use ARROWS to set Elution Volume to 230-1000 uL.
10. Press RESET to advance to “ELU nbr1.” Use ARROWS to set the Number of Elution Fractions to 1 or 2 for each separate well. Each elution fraction will be processed by 4 elution cycles.
11. Press RESET to display “YES SAVE.” Use ARROWS to select Save Program YES or NO. If YES, rewrite current program. If NO, run program, but do not save over saved program. (Note if enough memory is available, then pipette can save more than 1 program).
12. Press TRIGGER to display “PHY” which signifies ready to run. Pipette will beep.
13. Attach Pipette tip column(s) and submerge standless pipette with columns into the sample wells in the deep-well plate. Press TRIGGER. Pipette will display “CAP nbr1” and beep after processing the specified number of capture cycles. Pipette will display “CAP nbr2” if user specified more than 1 capture fraction. Move pipette tip columns to next capture well and press TRIGGER. Repeat until Pipette beeps and displays “Pur nbr1.”
14. Move standless pipette with columns into the wash wells in the deep-well plate. Press TRIGGER to run wash. Pipette will display “Pur nbr1” and beep after 2 flow cycles. If programmed to run additional washes, the pipette will display “pur nbr2.” Move the columns to the next wash well and press TRIGGER as guided. Pipette will beep when wash if finished.
15. When pipette displays “ELU nbr1” move the standless pipette with columns to the first elution well. Press TRIGGER to run elution. The pipette will beep after 2 elution cycles are finished. Pipette will display “ELU nbr2” if programmed for an additional elution aliquot. Move pipette with columns to the next elution well and press TRIGGER.
16a. Pipette will beep to signal the end of the final elution and will display “done”. Remove pipette with columns and dispose of columns. Press TRIGGER to begin the next set of purifications. Add new columns and go to step 13 to start the operation.
16b. Pipette will beep to signal the end of the final elution and will display “done”. Remove pipette with columns and dispose of columns. Press RESET to reprogram and begin a new purification method. Go to step 3 to start operation.
Pipette tip columns of silica can purify up to 10 μg of plasmid DNA. The purified plasmid is compatible with any downstream application including DNA sequencing, PCR amplification, transformation and restriction enzyme digestion.
Set Up Deep-Well Plates as Follows:
Plate 1
Row 1: Deep-well block row of preparation from step 4 of procedure below.
Row 2: Deep-well block row containing 300 uL of Resuspension buffer
Row 3: Deep-well block row containing 300 uL of Lysis buffer
Row 4: Deep-well block row containing 400 uL of Neutralizing buffer
Plate 2
Row 1: Deep-well block row containing 200 uL of Equilibration buffer
Row 2: Deep-well block row containing 200 uL of Wash 1 buffer
Row 3: Deep-well block row containing 200 uL of Wash 2 buffer
Row 4: Deep-well block row containing 200 uL of Elution buffer
Procedure:
Grow Cells:
1. Grow a single plasmid containing E. coli bacterial colony in 800 uL of 2×YT bacterial growth medium in 96-well 2 mL deep-well culture block.
2. Cover the plate with a gas permeable seal and shake at 300 rpm at 37° C. for 17.5 hours.
3. Pellet bacterial cultures by centrifuging culture plate at 2500×g for 10 minutes.
4. After centrifugation, remove the seal and invert the block to decant the media away from the cell pellets. Blot the inverted block on a paper towel to remove excess media.
Lyse the cells harboring the plasmid.
Add 250 uL of Re-suspension buffer to pellet bacterial culture using standard pipette and tips in normal manner.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover and variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth. Moreover, the fact that certain aspects of the invention are pointed out as preferred embodiments is not intended to in any way limit the invention to such preferred embodiments.
This application claims priority to and benefit of U.S. Provisional Patent Application No. 61/302,851 filed Feb. 9, 2010, the disclosure of which is incorporated herein by reference in their entirety for all purposes.
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