The Nanoliter Syringes

Following an informal collaboration with Affymax (Palo Alto, CA), Agilent Technologies (Palo Alto, CA), and Chem-Space Associates (Pittsburgh, PA), studying electrospray ionization 1 (ESI), the technology invented by Prof. John Fenn,2 a simple technology called induction-based fluidics (IBF) (Nanoliter, LLC, Henderson, NV)3 was invented and patented.3 IBF charges liquids, but without the adverse electrochemistry inherent in ESI. IBF has other merits as well. That said, the charging of liquids has been around for some time. Prof. Robert Millikan employed it to determine the charge on the electron, receiving the Nobel Prize in 1923,4 and many others have been charging liquids for numerous purposes, including printing, since 1951.5

In IBF, liquids are charged also, and that action allows the performance of many simple, useful tasks including flying nanoliter and microliter quantities of liquids nontouch to targets of all types, such as humans, plants, and animals; microscope slides; multiple-well plates; and scientific instruments.6–12 Through the presentation of the physics of IBF, it has been shown that unlike piezoelectric, sound, or any other technologies that are applied to transport liquids, IBF technology employs an electric field that can 1) kinetically launch drops to targets of all types, 2) dynamically direct the liquids in flight (a very desirable, i.e., required, trait for small volumes of liquids), and 3) count them on arrival. This simple technology has been called “elegant” by the director of R&D of a major mass spectrometry firm,13 and is revisited here along with new devices and applications (see Figure 1).

Figure 1 - Nanoliter liquid handling, including 22 nL, 3×, at 60× magnification to a matrix-assisted laser desorption ionization (MALDI) target; 40 nL flown to thumb; nL (varied) flown from a Gilson pipet (Middleton, WI); nL from syringe flown into a multiple-well plate; writing picoliter with pL; ca. 35 nL on dime; 8 channels, 50-nL dispensings; and the parallel LC/MALDI (Nanoliter, LLC), μm-sized droplets directly from LC columns.

Why nanoliters, again?

The nanoliter regime offers a number of obvious benefits over microliters and milliliters. These include 1) significant savings in expensive reagents; 2) major reduction in human exposure to toxic chemicals, allergens, agents, viruses, etc.; and 3) greatly reduced waste disposal costs. Because IBF has a massive dynamic range (μL to fL), it has a substantial application space. It is a useful laboratory tool that has wide application in, for example, simple sample dilution; MALDI biomarkers sample preparation; drug delivery; drug discovery; radiochemistry; homeland security and defense applications; forensics; the sampling of human beings; medical diagnostics sample preparation; and in the manufacture of unique chemical and other entities, i.e., electrets. These charged spheres of reagents can be manipulated with fields to effect unique liquid/solid handling tasks. (These were originally called nanoliter-sicles.) Finally, IBF allows nontouch dispensing in the microliter regime as well, for more classical assays, and also has interesting consumer applications.

Applications

Of course there are countless routine applications of the low-volume dispensing and aspirating technology, as given below. Applications include sample preparation for thin-layer chromatography (TLC), or even simple sample dilution or other sample handling tasks, which can in fact be highly parallel. This includes the recently patented ability to morph syringe, peristaltic, and other pumps into nanoliter dispensers. Moreover, IBF-based sample preparation provides the ability to save precious samples such as expensive enzymes or evidence in forensic settings, where disposable containers can be the dispenser, minimizing sample transfers and various loss mechanisms. IBF can dispense, i.e., literally fly, nanoliter quantities of highly viscous, complex liquids such as glycerin, some glues and slurries, and even heparinized whole human blood to targets like microscope slides, blotting media, and other targets over short distances (μm) to longer distances on the order of meters using simple devices. In certain settings, IBF can perform nanoliter or other dispensings from inexpensive, disposable patent-pending pipet tips (www. nanoliter.com). IBF applications demonstrating the wide and varied interest in low-volume dispensing are presented in Table 1.

With IBF, one can apply electric fields of different shape, energy, polarity, and intensity, and vary them in countless other ways. Because of this, one can morph existing traditional devices such as syringes, pipets, pumps of all types, LCs, solid-phase extraction (SPE) devices, and other instrument introduction tools into new, nontouch dispensing/sample preparation devices simply by appending IBF technology to existing devices. Thus, common tools that are deployed in laboratories and factories worldwide can take on useful new attributes that can facilitate the transport and treatment of liquids from the macro to micro to nano world and back, often extending the dynamic range of devices by 1, 2, or 3 orders of magnitude. This property, the ability to establish “world to nano” connectivity or to provide a path from the macro, micro, and nano world, may prove to be one of the most useful aspects of IBF. In fact, one can place liquids onto humans for drug delivery or sample them using a patent-pending process that is nondispersive (see Figure 2).

Figure 2 - Liquid being pulled into a cone jet to the target from a human in a nondispersive manner.

Implementation

Recognizing that the technology was originally introduced at a very bad time historically, i.e., September 2001, one must wonder why the nanoliter regime has not been widely adopted given all of its potential. One possible reason was the lack of inexpensive, commercially available tools. To, in part, remedy this, the nanoliter, microliter syringe was invented. This simple device can morph any common microliter syringe into a nontouch nanoliter or microliter dispenser (Nanoliter, LLC). In the simplest applications, IBF technology can now deliver cost-effective, highly accurate, precise, routine, economical nanoliter liquid handling to laboratories worldwide.

Figure 3 - The nanoliter, microliter syringe with ca. 40-nL dispensings of MALDI (α-cyano-4-hydroxycinnamic acid, CHCA) matrix on a MALDI Applied Biosystems (Foster City, CA) target. Circles are ca. 1 mm. Accurate volumetric and spatial sample placement can improve MALDI results by increasing sample homogeneity and by spatially concentrating analyte in spots the size of the laser beam, as compared to larger volumes of the dried droplet method that results in shooting target locales with no sample, generating prima facie-only noise.

The nanoliter, microliter syringe (Figure 3) can morph standard microliter syringes into a nontouch nanoliter or microliter dispenser once placed in the chamber. The same device in 8- or N-channel mode can place liquids into multiple-well plates or on surfaces with the volumetric and spatial accuracy needed to improve MALDI quantitative and qualitative analysis when coupled with the programmable controller (Nanoliter, LLC). This control can be seen in a video at www.nanoliter.com and in Figure 4, which shows the liquids at the bottom of the multiplewell plate instead of outside, or on the sides of the wells, unlike other technologies that do not dynamically direct the liquid.

Figure 4 - Nanoliters flown into a multiple-well plate, the patented energy controller, and an IBF spray-based deposition tool based purely on induction.

It is important to realize that IBF can be used to place samples anywhere, flying them in simple or complex trajectories from humans (not just to them), to instruments, up to targets (i.e., aspirating them), down, left, or right, in a manner completely analogous to how gas phase ions are flown in a mass spectrometer as given by SIMION™ (the software developed by David Dahl for ion and electron optics simulation, see www.simion.com). Unlike gas phase ions in mass spectrometers, however, IBF-based liquid trajectories must include other forces.

To understand IBF,5 consider the physics of a flowing laminar system. The liquid volume passing through a tube is given by the Hagen Poiseuille equation. (IBF does not need hybrid systems, and the flow can be purely electrokinetic, but that is beyond the scope of this paper.) The volume of fluid (V) that flows down a small-diameter capillary tube per unit of time (t) is proportional to the radius of the tube (r), the pressure pushing the fluid down the tube (P), the length of the tube (l), and the viscosity of the fluid (η). Note, V is linear in t.

V = ((πr4P)/8ηl)t

Now, if we grow a drop on a capillary under these conditions, we can then charge (q) the drop using an electric field E to energize it. Upon charging, the ions rapidly migrate to the surface of the drop. Now that the liquid is charged, it can experience the electrical force (qE) imparted by induction, similar to the manner in which gas phase ions experience the qE force in mass spectrometers. Since electric fields can be rapidly toggled on and off with high accuracy and precision, the forces on the liquid drops can be changed rapidly and accurately as well.

F = qE

Because F is a vector, we can direct the drop if we shape the field. Thus, for a charged drop with initial value, q0, which depends on well-known solution specifics,

q = q0 e(–t/λ)

we have a relaxation time, λ, where λ= (ε0εr/κ), εis the dielectric constant of free space, εr is the relative permittivity, κ is the solution conductivity, t is time, and q is defined.

Now, a charged liquid drop in an electric field not only can experience the qE force, but experiences different forces as well in the atmosphere in x, y, and z space, depending on the specifics of the system, as stated previously. Using standard, well-known physics, Newton’s Second law, we can equate the forces (electric, drag, buoyancy, gravity, and coulombic) acting on a drop to those acting in the direction, x, as:

Fx = m (ax) = m (dvx/dt) = Felec + Fdrag + Fbuoy + Fgrav + Fcoul

Force equations can also be written for the y and z coordinates; therefore, with accurate model equations for Fy and Fz, we can actually calculate the trajectories of the drops (distances of travel, d) at any time, t, knowing that Vx = dx/t, Vy = dy/t, Vz = dx/t, and the initial position of the drop, and that V2 = Vx2 + Vy2 + Vz2, but that discussion is beyond the scope of this paper.

Simple observations of charged liquids

Figure 5 - Visit www.nanoliter.com to see this drop fly up, i.e., aspirate to a thumb; ca. 7.5, 15, 22.5, 45, and 90 nL, estimated using stepper-motor digital readout in this case.

One aspect of IBF is the sometimes counterintuitive ways that charged matter behaves. For example, when a liquid is charged to some value, q, in a field of strength, E, with gravity acting in the opposite direction, the drop can be aspirated, i.e., it flies up (www.nanoliter.com). In fact, it has been known for many years that one can literally levitate drops in laboratories. However, with IBF and this new tool, the technology can be applied to such activities as depositing the liquids onto targets for proteomics biomarkers/cancer disease diagnostics, drug delivery, placing samples into scientific instruments, simple dilution, conserving precious reagents, and countless other purposes (see Figure 5).