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WHY NANOLITERS ? Copyright 2004 Andrew D. Sauter, Jr Abstract: I discuss why using lower volumes of liquids make sense in the cancer diagnostics, laboratory and other market space. I also discuss how, why and where a new approach, induction based fluidics, affords new functionality across a wide flow range. I parallel IBF's use of electric induction in liquid dispensing with electric induction employed in mass spectrometers to "dispense" gas phase ions. Examples shown herein, at the links and in the references show that IBF affords new liquid handling and material manufacturing capabilities in both the macro and the microfluidic world. Transporting liquids is among the most frequently performed task in laboratories of all types. This “function” is also performed to manufacture liquid products (e.g., drug solutions) and it is required at locales of health care. When small quantities of liquids, e.g., nanoliters*(nL’s), are employed instead of larger volumes, it is obvious that the cost of reagents used can be reduced. Such cost savings can be significant in many areas reducing the costs of many assays including: matrix assisted laser desorption time of flight (MALDI TOF) sample preparation for cancer or peptide pattern detection(1) ; in DNA/RNA testing; in the area of nuclear, chemical and biological detector development; in HTS drug discovery and development work and many other application/markets. Reducing the amount of liquid employed in a protocol from 50 uL to 50 nL saves a factor of 1000 in reagent costs at 100 percent utilization. Recognizing that the utilization rate might be less than 100 % and that other costs might be incurred, saving reagents makes economical, and technical sense yielding fast payback given such multiples. This is particularly true when smaller volumes of expensive reagents (e.g.,rare chemicals; enzymes, stable isotopically labeled or radio labeled analytes), are employed as the savings add up fast, as you purchase less in the first place. Of course, adopting the nanoliter or picoliter regime for sample dispensing, or other simple functions (dilution, desalting, parallel LC) must be both cost effective and convenient for wide acceptance. Another obvious reason for employing smaller quantities of liquids (i.e., nLs) in the lab or factory is that one can reduce exposure of humans to toxic or hazardous materials, as it mitigates liabilities and danger. For example, using nanoliter quantities of materials in teaching non-major labs, could increase safety, and savings as it reduces other expenses for lab equipment (e.g., facilitating smaller more portable hoods that save bench space, utility costs, etc.). Clearly, reducing that amount of material employed in all experiments using simple advances in microfluidics, is possible, and desirable. In addition to significant reagent savings and enhanced safety, using smaller quantities of liquids reduces wastes and waste disposal costs. In fact, for many labs that employ LC in the USA, much of Big Pharma, waste disposal costs can be greater than the solvents themselves! Obviously, throwing away lesser amounts of very expensive material that becomese a regulated hazardous waste is desirable and saves significant funds as well. In addition to the aforementioned, the ability to manipulated nanoliters, picoliters and microliters using our accessible technology (i.e., Induction Based Fluidics,IBF) allows one to perform new functions. For example, IBF can serve to join the macro and micro world by placing samples into/onto LabChips or scientific instruments(2). Alternatively, as indicated at proteomicssurf.com. in the field of cancer diagnostics (1) one can cleanup and place samples for MALDI –TOF mass spectrometry (MS) using traditional UV lasers or as in the newer, very exciting aqueous matrix, atmospheric pressure infrared IR MALDI TOF MS (3). Additionally, as discussed below, one can make new entities, nanoliter-sicles that can be "picked up" and manipulated, as if they had "handles." For all of the above reasons and for many more, it is natural to expect that devices that can transport nanoliters could have positive impact in the areas of medical diagnostics, in modern laboratories and in other areas including the packaging of drugs and reagents. In non laboratory markets, the ability to reliably and inexpensively produce low flow rates, might allow prescriptions to be prepared at home, fresh using less filler or as a part of diagnostic in-home health care devices. So the simple ability to create and to manipulate nanoliter quantities of liquids has significant economical, technical and practical benefit. As such, a part of the answer to the question first asked in Photonics Spectra (4 ), “How will we handle nanoliters ?” includes we hope induction based fluidics (IBF) for reasons discussed below. HOW NANOLITERS ? We have been discussing nanoliter transport using IBF for some time (5). IBF unlike electrospray (for which Professor John Fenn shared the Nobel Prize in Chemistry (6) ) is another electrokinetic fluidic transport tool which does not have to spray, although it can. IBF employs an electric field to deposit energy directly into a liquid by electric induction, through the containers. That is the electric field directly couples with the liquid (or liquids!). Placing a tube or many tubes containing a liquid/s into such a field can make common devices like pipette tips (e.g., Zip Tips (TM)), capillaries of all types, and other lumen, transport the liquids electrokinetically often with no modification of the expendable (7). Such capabilities could afford many benefits in forensic analysis, as samples could be saved for later replicate analysis, because when using IBF, the container can be the dispenser (7). A very simple generalized schematic for IBF is given in Figure 1 below, and elsewhere (8 ). As simple as this “schematic” is, there’s more than meets the eye. For example, 1. The containers can have different shapes, 2. They can be made of different materials., 3 They might containing different liquids and 4. The containers might have different substrates in them, such as filters, solid phase extraction (SPE) media or LC phases. Now, if each of the four characteristics has only ten possible variants, the simple diagram given as Figure 1. could produce 10^4 possible configurations of a macro/microfluidic device. Actually that turns out to be an extremely conservative number of possible IBF configurations. INDUCTION BASED FLUIDICS
Figure 1. Electric field (arrows) inductively coupling energy into five containers having different cross sections and where the number of liquids, energy, number of channels and dispense time might be varied.. In Figure 2, example output is presented where the number of channels, the channel cross section, the dispense time, the dispense energy, the number of channels, the number of liquids and the number of reservoirs were change to produce the different dispensing patterns. Typically in such work the area for a given dispense (Note all examples are not to the same scale.), is converted to pixels assuming a proportionality to the dispensed volume. Then this is correlated with dispense time at a given energy. Of course, we can construct a calibration curves to verify our assumptions that this correlation process is accurate by demonstrating a zero intercept and linearity. (Note in Figure 2, the nanoliter and microliter examples were typically dispensed in a few seconds, where as the picoliter examples were acquired over approximately 120 seconds. Other, inexpensive configurations can produce low pL/sec flows over short times.) Below we present additional output for microliter, nanoliter and picoliter dispensings. The two microliter dispenses shows four channels using same liquid (all examples use 50 % ethanol and water with food dye). The two channel example shows five dispenses whose dispense time went from 0.5 sec smallest to 2.5 sec and had different cross sections, and hence produced different volumes. In the nanoliter area the two leftmost examples show a four channel head with the same four liquids where all variables were the same except the dispense time. The smaller the blots, the shorter the time, the smaller volume. (Note that the visual quality of the dispense is influenced by the receiving surfaces, paper in this case.) In the next nanoliter example in the center, only the cross section was varied using a three channel system and the dispense energy and the time was the same. This, of course produces different volumes or flow rates concurrently with one “pump.” The second rightmost example, shows a single channel, five dispense at four levels to produce a calibration curve. The last or right most nanoliter dispense shows output generated from a microtiter plate washer (whose cost was $3k) that I readily morphed into a nanoliter dispenser where in this example, all parameters were the same for the three channels. The final nanoliter example located under all previous nanoliter dispensings shows twelve different liquids dispensed where all parameters (i.e., energy, time, cross section) were the same, except that twelve different liquids were used. Note that the picoliter dispensings shown at the bottom of Figure 2 are single channel examples acquired in 120 seconds producing flows in the low picoliter per second range. (Note other arrangements produce low pL/sec flows as shown in Figure 3.) So the very simple Figure 1 representation of such a dispenser can produce countless configurations, by varying the shape, the material, the contents and the number of the lumen and other variables including: the energy; time and the lumen contents. In fact literally millions of configurations and functions can be derived from IBF devices. This is especially true when you consider that one might want to layer or stack various electric induction driven devices. Examples of Parallel Dispensings of uL, nL and pL
Figure 2 Examples of different dispensings that can be accomplished with IFB. Electric induction generates the force/s on liquids in a manner similar to that employed in mass spectrometers (MS) to transport gas phase ions. Electric induction is employed as the accelerating potential and focusing elements (lenses potentials) in an ion trap or , for example in a triple stage quadrupole TSQ MS. An extremely interesting aspect of this force generating principle, is that electric induction has an enormous dynamic range of operation. For example, twenty-two years ago, it was shown that electrons and positrons could be "dispensed” (one at a time.) from an ion trap, (obviously) in the “gas” phase (9 ).Of course in terms of mass that is 10^-28 grams per gas phase “dispense.” At nanoliter.com and picoliter.com we show a wide range of weight or volume of IBF based dispensings at pico, nano and microliter levels, often in parallel. In one case five, 48 channel,48 different liquids 100 nL parallel dispenses is shown in twelve seconds. Of course, if one dispenses enough 100 nL’s, one can accumulate uLs which of course are 10^-3 grams or milligrams. Hence, electric induction can contain and dispense matter as described above and herein over 25 orders of magnitude. So, an answer to the question: ”How Nanoliters?” is IBF. IBF, operates uniquely, in parallel, across an enormous dynamic range using as little as one supply of energy, per N channels without moving parts, adverse electrochemistry and significant joule heating. Another attribute of IBF is that the force generated can be changed rapidly. For example, for gas phase ions in a TSQ MS, ions of both polarities are transmitted in alternate scans by changing the polarity and voltages of source, lenses and rod offset potentials in nanoseconds. Hence, the device passes negative ions (a negative ion pipe) in one scan and in the next instant with polarity/voltage changes in accelerating, offset and lens potentials, it transmits positive ions (becoming a positive ion pipe), rapidly. That is another way to say that the vector quantity, the electric field strength (E) rises and collapses very fast in vacuo. Hence, as F=qE the force (F) on charged entities (e.g., liquids) in electric fields of strength (E) can be applied/relaxed rapidly. Considering liquids, we can expect that rapid and precise activation/deactivation can help energize solutions quickly. Since for a fixed system per Figure 1 where time is directly related to the dispense volume (10,11,12,13,14 ), induction allows us to change the force applied to a liquid very quickly and reproducibly. This bodes well for dispensing accuracy and speed. Moreover in IBF based devices, liquids can be “flown” up, down, left and right, in a manner similar to gas phase ions in a MS system (See video at picoliter.com,15.). Therefore an IBF system, the liquids of course, have mass and can have charge. These liquid constellations of energy can emulate gas phase ion behavior. By that I mean that such small quantities of liquids can be transported to different locales with different trajectories, not unlike gas phase ions in a mass spectrometer.
Figure 3. Writing the word “picoliters” with pL’s. The approximate 1.0 uL blot was dispensed from a standard microliter syringe. Mass spectroscopists have been "dispensing" nL's and pL's for years (6, 16), although IBF is a different, new approach. WHEN & WHERE NANOLITERS ? PROTEOMICS, AS WELL AS, NUCLEAR, CHEMICAL & BIOLOGICAL APPLICATIONS ,RIGHT NOW ! As to dispensing many vendors are now beginning to claim nanoliter capabilities. To my knowledge, most if not all, are at higher levels hundreds of nanoliters which we assert are largely low microliters, and performance at such levels is not generally rigorously established. That stated, activity is very strong and answering the question “When nanoliters?” I believe that the answer is: “Right now, and particularly in a few areas, one being cancer diagnostics." Recently, NCI and other governmental organizations, MS instrument firms and companies involved in genomics and proteomics have been reporting major advances in the detection of cancer. A major reason for this is that "protein patterns" identified by MALDI TOF have been shown to diagnose ovarian, prostrate, lung, brain, colon, pancreatic and other cancers and other protein based diseases (e.g., Alzheimers). Since many tests can now use serum and very low biomarker concentrations can be detected, cancer and other diseases can be identified without surgery, detected early, which bodes extremely well for treatment. For example, recent work has shown that Stage 1 ovarian cancer can be detected early in serum with 100 % accuracy. Since treatment success can be greater than 90 percent for this cancer, at that stage and since in general, early detection bodes well for treatment (potentially prior to the creation of tumors, ect.), our patented technology which can cleanup and tightly place samples for MALDI TOFanalysis has a major massive, "new" and important application. (Suggested reading to verify these discoveries and implications can be found at http://www.proteomicssurf.com. Also, see Analyical Chemistry, See Novartis article, May 1, 2003.). We are very excited about that application. That stated, the full impact of nL’s in application space is actually just beginning, as there are so many. “When will we feel the full impact of Nanoliters?” That is occuring now in proteomics. It is also occuring in NCB work and it will also occur in other applications/markets when it is apparent that the lower cost of solvents, improved health benefits, reduced liabilities and lower waste disposal costs are understood by many. Then, when affordable, rugged, simple instrumentation is available, nanoliters will be ubiquitous. “Where Nanoliters?” will, I believe, be in most labs worldwide for chip based and non-chip based assays and in devices performing more mundane applications. It is anticipate that IBF will play a part as it has been shown able to make Zip Tips "Electric", dilute samples over many orders of magnitude in one operation without generating intermediate solutions i.e., that create costly hazardous wastes, and make unique entities such as nano and picoliter-sicles. UNIQUE NANOLITERS OR NANOLITERS-SICLES ! Dispensing is just a small part of IBF. Patented applications (8 ) include: parallel LC; filtration; SPE; instrument introduction and serially parallel derivatives thereof. Interestingly, small volumes of liquids can be created in an IBF driven device creating what I have called "nanoliter-sicles" or "picoliter-sicles". This process is shown picoliter.com. These charged functionalized spheres can be made in a second or two such that they can be “aspirated” (i.e., transported) with a metal rod and other devices, (,i.e., easily physically handled.). Video at picoliter.com demonstrates how such small drops can be dispensed, and frozen allowing one to overcome the sample handling problem of “adhesion.” This is a major issue in low volume dispensing that IBF and Nanoliter can solved in this manner. I have also begun to do plate to plate transfers with nanoliter-sicles, as well (potentially lowering robotic costs) as small volumes can be rolled or flipped into N {N=96, etc.} wells at a time like spherical flap jacks, and one robotic move. In point of fact, the analogy of treating IBF liquids like gas phase ions is while, explicitly incorrect, useful. Imagine how protocols might change if one could accurately and routinely dispense AND manipulate 23 nL +/-< 5 % at one SD (7) . IBF has been shown to make Zip Tips “electric” dropping digest to set locales that could facilitate MALDI-TOF as discussed above making it easier to find the “sweet” spot and acquiring good spectra (7). Using IBF and an appropriate stylus, (See figure 3) writing the word “picoliters” with “picoliters” can be accomplished. Such devices have many simple uses just like the microliter syringe, e.g., making standards, simple dilution or parallel solution/standard production. WHO NANOLITERS ? Nanoliter hopes to play a part in this transition. We seek to license the technology, as we continue development new technology. Others are forwarding their state of the nL technology, at many firms worldwide. As large groups prove the nanoliter market, IBF and very powerful pending derivatives, will evolve into often simple devices found in many labs in the world, in factories and potentially in the home, we hope. Simple related technology has to a certain extent rewritten the economics of other basic human enterprises, such as printing, so such a bold statements may not be hyperbole.
Alpha Prototype, Nominated For Best New Instrument at Pittcon. SUMMARY A simple approach, not at the edge of physics (17), that transports liquids for useful, sometimes mundane, but important purposes in the macro/micro world has been discussed. The device can be polyfunctional transporting small quantities of liquids onto media or into receivers (including scientific instruments) for use in the laboratory, and potentially in the factory or at home. A very new application allows the device to feed MALDI TOF instruments whose ability to detect "peptide patterns" is revolutionizing the early detection of many types of cancer.Verify at http://www.proteomicssurf.com.). The device can also play a role in the development of NCB devices used to detect agents in counter terrorism test development, in the Homeland Security market. Since the simple device, can place nL, uL and pL quantities of liquids onto media and into receivers in parallel without or with touching it has countless applications. Unlike older complicated techniques that employ pistons, vibrating polymers/ceramics or other force generating principles (e.g., syringes) the new device uses an electric field as a source of energy in a manner that requires only one source of power to move, N channels of liquids. Because older techniques would require many sources of energy or more complicated designs this patented approach has excellent economics in many markets. A significant figure of merit of IBF is that the device can transport liquids across a very wide dynamic range (approximately 9 orders of magnitude from uL/sec to low pL/sec). When coupled with filters and other devices (LC Phases) multiple functions can be performed, very inexpensively on a per channel basis (8). Hence, the new technique (actually techniques) has useful physical and other attributes including high precision and accuracy, due to it's simplicity and the elegance of using an electric field to energize liquids in containers. Such simplicity, low costs, and many applications bodes well we anticipate for IBF, as we bridge the macro and the microfluidic worlds. ____________________ * I ignore much larger induction based (electric and magnetic) processes, including solar flares inductively creating massive currents to our electrical grids, atmospheric and astronomical phenomenon. References 1. Broch, A. et al, Analytical Chemistry, V 75, pg 2309, 2003. 2. Go to http://www.nanoliter.com. Examine the pictures, video and other areas of the site. 3. Doroshenko,V.M, et al, J.Am. Soc. Mass Spectrom., 2002,13,354-361. 4. Drollette,D. Adventures in Drug Discovery. Photonics Spectra 1999:86-95. 5. A. D. Sauter, W. Fitch, J. Chakel, A. Affel, R. Willoughby and E. Sheenan," Approaches For Estimating ESI/MS/MS Relative Response, presented at Pittcon 97, New Orleans, LA, March 1997. 6. Fenn JB, et al, Science 1989,246:64. 7. A. D. Sauter Jr. and A. D. Sauter III, “Electric" Zip Tips For MALDI And Other Applications, Journal for the Association of Laboratory Automation, May-June 2002. 8. At http://www.uspto.gov, search USA Patent No. 6,149,815. 9. P. Ekstrom and D. Wineland, Scientific American 243, "Ion Trapping:", Scientific American 243 #2, 105 (August, 1980). 10. A. D.Sauter, Jr. and A. D. Sauter III, “Nanoliter’s Onto Media, Use Of Electric Induction” American Laboratory, October 2001. 11. Government Rating Scheme for Industry Briefing, US Army Soldier & Biological Chemical Command, Edgewood Arsenal, October 2000. Available from author. 12. A. D. Sauter Jr. and A. D. Sauter III, "Nanoliter-sicles" presented at Pittcon 2002, New Orleans,LA. 13.. A.D. Sauter Jr. ”Flying Nano, Micro and Picoliters Using Electric Induction”; invited presentation at Microfluidics 2002, San Francisco Sept 20. 14. A.D. Sauter Jr., D. Eastwood, and R. Willoughby, White Paper:” Proposed Proof Of Concept Study: A "Clutter" Filter or A Real Time, Electric Induction-Based Air Sample Cleanup Device Assisting The Detection of Bio-agents & Potentially Various Bio-agent Detectors.” Released To USDOD, April 2002. 15. Go to http://www.picoliter.com 16 R. Caprioli, R. , Editor in Chief, J Mass Spectrom, JMS Special Features 1998-2000. 17. See Scientific American, Special Edition; "At The Edge of Physics;"Vol 13, No. 1., 2003
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