How to make glass microfluidics device? chemical wet etching and room temperature bonding

[First part of this post is here where I wrote step by step procedure for mask design and photolithography.]

After completing the photolithographic procedure, we use two different solutions for selectively removing chromium and glass from the channel network on the bottom substrate. 

Chemical wet etching solutions. On left is BOE with temperature control
and on right is Chrom etchant. Middle one is DI water.

At first, chromium layer from channel network is removed by immersing the substrate plate into a Teflon coated jar containing chromium etchant solution for about 15 min. One should note that leaving substrate plate in this solution for long time (hours and hours-this happens when you forget to remove it) could eat away even the photoresist layer from the entire plate. We buy the chrome etchant from Transene Company, INC, MA, USA and according to manufacturer specification the solution contains ceric sulfate (5-10%), nitric acid (5-10%), sulfuric Acid (1-5%), water (~ 75%). The entire substrate is then washed with DI water and dried by blowing N₂ gas. At this point one can clearly see through the substrate in channel patterning region.

Cartoon depicting the removal of photoresist and chromium layer from channel pattern

After this step, we cut the substrate into individual chips (1" x 2") using a glass-cutter (altogether 8 chips from one 8” x 8” substrate). Then, put extra layer of photoresist, dry in oven (80 ^o C), take out from the oven, and let it cool.

The substrate plate-individual chip is then immersed into a 1:10 buffered oxide etchant (BOE) solution bought from Transene Company, INC, MA, USA. According to the company specifications, this solution contains a mixture of hydrofluoric (HF) (1-25%), ammonium fluoride (NH₄F) (2-40%), distilled water. Hydrofluoric acid is a hazardous chemical, therefore must be handled with extra precaution. The BOE solution is continuously stirred at the rate of 120 rpm using a magnetic stirrer and the temperature is set at 55 ^oC. The substrate plate is removed from the BOE solution at a certain interval of time ~10-15 min (depending on the etching rate of BOE and channel depth desire), washed with DI water, and is dried with N₂. With our settings, etching rate is ~0.5 micron per minute. 

However, this rate can be manipulated based on your need. 

Cartoon showing the substrate plate after removing glass material.
Remember the glass etching is in all direction (not like in this cartoon)

The depth of the channel is measured using a XP series stylus profiler (Ambios Technology, CA, USA). This step is repeated until one gets desired channel depth.  

Profiler

Once desired channel depth is obtained, ~1 mm diameter access holes were punched at the channel terminals using a microabrasive power blasting system (Vaniman) by blowing sand particles from back side of the substrate. The miroabrasive system utilizes mechanical erosion on the substrate by bombarding with high-kinetic energy sand particles and results in conically shaped holes. This step is followed by removing the photoresist and chromium layer on remaining parts of the bottom substrate by using acetone and chromium etchant, respectively. 

Sandblaster 

Bonding two glass plates: The etched channels in the bottom substrate were sealed using cover plate. The two plates (bottom substrate and cover plate) were cleaned and brought together in a beaker containing DI water for bonding. They were removed from the beaker together with a tweezer, water was removed by gently pressing the plates with hand in paper towel, and then put under couple of heavy books for ~2 hrs. Then the bonded chip was kept in an oven at 80 ^oC for ~1hrs to strengthen the bonding between two glass plates. Bonding is a crucial step to create closed fluidic microchip networks. 

The bonding strength between two surfaces is proportional to the density of individual chemical bonds established between the two plates. Our bonding method is simple and does not require clean room facilities, programmed high-temperature furnaces, pressurized water sources, and adhesives. The bonding process can be completed in ~3 hrs. One of the most important factors affecting successful bonding of planner glass chips is the cleanliness of the bonding surfaces of glass substrates. Unsuccessful bonding events are often associated with solid particles or organic material remaining on the glass surfaces, for example, dust, residual photoresist, chromium or glass particles etc., prior to bonding. Some other critical factors for glass bonding are flatness of the glass plates and chip area.

ELISA with 100 fold improvement in detection limit & 100 fold less sample volume

My first paper on microfluidic work has been published in Analitica Chimica Acta. This was the first project I worked on for my PhD work (actually the very first one didn't work. That was my advisor's crazy idea he wanted me to give a try.  We abandoned that later on). 

In this article, we have demonstrated a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4- methylumbelliferone) in free solution. 

Highly sensitive analytical techniques are required to estimate small amounts of disease markers (e.g., antibodies/antigens) in bodily fluids in order to detect the onset of dangerous diseases like cancers at their early stages. Here is the abstract of this paper. 

no-instrument measurement in paper-based device

Paper-based devices (PBDs) have emerged as a new class of inexpensive devices with potential use in medical diagnostics, environmental applications etc. They are not only inexpensive but also proven to be sensitive, quantitative, and reliable in many applications.

The colorimetric detection is one of the popular methods of quantitation of analytes in PBDs. Color developing reagents are used on the micro-reaction chambers on paper. The color developing reagent then reacts with the analyte of interest when the later is applied specifically and quantitatively. Simple and widely available instrumentations like a office scanner or a camera (could be a mobile phone camera) are used to capture the color which is then quantitated by measuring the signal using image processing software.  If you don't have camera and/ or image processing software, don't worry. Scientists from Colorado State University have developed even a simpler method to quantitate analytes. This interesting work has recently been published in Lab on a Chip Journal.

The new technique relies on the length of the color developed. They call this as a simple distance-based detection. You just need a ruler to quantitate the analyte concentration: no camera, no scanner, no software.

How does this work?

1.

First of all create a paper-based device. In their work, they have used wax printing to make PBDs. Their design has a straight channel, like a thermometer. There is one circular reservoir at one end. This reservoir is used for sample addition and/or transfer of analyte to the detection zone (long conduit).

2.



Colorimetric detection reagents are deposited along the flow channel. They tested two different methods for this purpose: spray application or pipetting. Once the reagents are dry, the device is ready to use.

3.

Sample solution in water is then added to the sample reservoir which flows into the detection/flow channel. The analyte reacts with reagent and develops color. Once all of the analyte is consumed, the color development stops. Analyte quantification is then done by measuring the length of the colored region in the detection zone by a ruler.

Authors of this paper have demonstrated their technique by measuring three different analytes: nickel, reduced glutathion, and glucose.

This new measurement technique is claimed to be accurate and sensitive compared to conventional methods.

In this figure, you can see that higher the concentration of analyte-the longer is the color developed region in detection zone.

shallow region on microchannel for better separation

CZE technique, introduced in the 1960s, is used to separate charged analytes under the influence of an electric field. Charged analytes are separated on the basis of their size to charge ratio. Some of the applications of CZE include separation of DNA (e.g, in DNA fingerprinting), separation of proteins, analysis of basic drugs and related substances in pharmaceutical analysis etc.

Researchers from University of Wyoming have demonstrated that "shallow region" in a microfluidic separation duct enhances the resolving power of capillary zone electrophoresis (CZE). They have shown a 8 fold increase in resolving power while separating a mixture of amino acids utilizing this technique. 

Simple microfluidic device to have better separation in CZE
as described by Xia and dutta (copied from Anal Chem)

In their recent article published in Analytical Chemistry, Prof Dutta and his student Ling used pressure driven back flow (counter flow to electroosmotic flow in the microchannel) to achieve their goal of high resolution CZE. This back flow was produced by a ~0.5 um deep shallow region. When electric field is applied on the separation channel, a mismatch in electroosmotic flow generates pressure driven back flow. Even though this back flow introduces some band broadening, overall increase in resolving power was better than a device without shallow region.

Authors claim to have following advantages in their device:

1. This device omits the use of an external pump for pressure generation simplifying its fabrication and operation;

2. It can readily be integrated into any detection and/or downstream analysis method; and 

3. It is suitable for further miniaturization of the CZE technique to the submicrometer length scale.

link of article dx.doi.org/10.1021/ac302530y

Analyte preconcentration results in detecting lower concentrations of cancer marker

It is important to detect lower concentrations of disease (like various cancers) markers in human bodily fluids reliably. The ability to detect lower analyte concentration would allow us to identify the onset of diseases in their early stages and start treatment process.

In an effort to make a device that could measure lower concentrations of protein, researchers of Dutta  group at the University of Wyoming have developed a microfluidic device in which the analyte protein molecules are preconcentrated infront of membrane inside the channel. Enzyme Linked Immunosorbent Assay (ELISA) was performed on the same channel and the signal generated from the enzyme reaction was increased by a factor of 200.

In their current work reported, they have shown a reduction in the smallest detectable concentration of the tumor marker CA 199-9 and Blue tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without preconcentration. This led to the improvement in the lowest analyte concentration detectable (LOD) by a factor of 20. Their device only required ~5 uL analyte and other solutions to complete the assay in contrast to conventional microtiter plate based assayed (~100uL).

There are several methods to amplify the ELISA signal and some of them involve preconcentration of enzyme reaction product (e.g resorufin) molecules. Dutta groups new method is unique in terms of preconcentrating target analyte-proteins. They used photopolymerized semipermeable membrane fabricated inside bonded glass microchip for preconcentrating analytes.

Details of this work has been published recently in Analytical Chemistry journal.

Microfluidics for STD diagnostics in the developing world #SamualSia

Dr. Samuel Sia, Professor of Biomedical Engineering at Columbia University, talking on poin of care diagnostic devices for detecting disease markers during Cla2010 Conference on Global Health Diagnostics "GHDx Innovations Summit: Translating Ideas into Impact".

Instruments/equipments in a microfluidic laboratory

I am presenting the instruments/facilities that we use in our microfluidic/nanofluidics instrumentation laboratory. We primarily fabricate glass based microfluidic devices ourselves and use them for different biological applications.

1. We have a basic photolithography setup that includes a dark room, UV light source and developing solution.

2. Then we perform wet chemical etching to make channel networks on the substrate glass plate using BOE (buffered oxide etchant) which is an appropriate mixture of HF and NH4F.

Chemical wet etching setup

The BOE solution is heated to certain temp to fasten the etching rate and stirred continuously to have uniform etching. The solution on the right is Chromium etchant which removes chromium layer from the substrate plate.

3. Sand blaster is used to make holes to have access to the channels. It uses 25-50 micron  sand particles which are bombarded on the surface of glass substrate. It makes holes of ~1mm diameter.

Sand blaster to make holes

Stochastic protein expression in individual cells at the single molecule level

(This was a project report for a microfluidic class written by Ravi Peesera.)

Introduction

Microfluidics is a prominent modern technology which deals with the manipulation of fluids in very small quantities, and has a very large number of potential applications in many fields of science.^2,3 In the field of analysis, microfluidics offers distinct advantages in conferring the ability to handle minute quantities of samples, when coupled with powerful analytical techniques opens up a vast number of applications. Single molecule detection methods are the emerging technologies which carry out measurements at single molecule level, which remove the discrepancies in bulk, ensemble average sample measurements. Microfluidics, with small sample handling capability, makes single molecule measurements possible and further makes the experiments highly designable, in allowing varied designs of miniaturized microfluidic chip systems.

The publication “Stochastic protein expression in individual cells at the single molecule level”¹ is a relevant example of measurements carried out with the combination of microfluidics and single molecule technology, in understanding the events of protein production. The transcription of DNA to messenger RNA and the translation to protein synthesis that follows occurs in a stochastic way in living cells. This randomness makes the development of assays to identify processes during the protein production difficult on a large ensemble of cells, due to the lack of synchronization of these events between cells, thus necessitating the use of single cell level measurements to understand these processes correctly.

β-galactosidase (β-gal) is a hydrolase enzyme that catalyzes the hydrolysis of biologically bound galactosides into galactose monosaccharides in living systems.⁴ Further, β-gal has been shown in various studies to be the standard reporter for gene expression in prokaryotic and eukaryotic cells. In 1961, Rotman and coworkers demonstrated that β-gal is active only as a tetramer and that a single molecule of β-gal can produce a large number of fluorescent product molecules when treated with the synthetic FDG molecule shown in the figure below.⁵ As shown in the figure below, β-gal reacts with the synthetic FDG, which contains two galactose units, cleaves both of them and liberates two molecules of galactose and one molecule of the fluorescent fluoroscein molecule. By virtue of the enzymatic activity β-gal reacts with a large number of FDG molecules, breaks them down by hydrolysis, thus liberating a large number of the fluoroscein molecules. This leads to an accumulation of these fluorescent molecules in the cell which would serve as a marker for the presence and thus the expression of β-gal, which in turn pinpoints to events in protein synthesis. Thus the accurate measurement of this amplified fluorescence in the cell at the single molecule level is crucial to development of an assay to understand this process in real time.

This exiting design of the experiment however has a limitation biologically. After the hydrolysis of FDG’s by the β-gal expressed during protein production, the resulting foreign organic fluoroscein molecules are acted upon by the cellular mechanism and are pumped out of the cell cytoplasm by the efflux pumps on the cell membrane, actively and efficiently. The pumped out fluoroscien molecules diffuse away rapidly into the extracellular region, and so the amplification of the fluorescent signal gained in the cell is lost. So in order to suppress the loss of fluorescence signal, arresting the diffusion process and the localizing it to an area close to the cell is crucial for the measurement of this signal without loss of the enzymatic β-gal amplification.

Theory and Principles

Long Cai and coworkers at Harvard chose to address this problems by using microfluidics to develop an assay to measure the fluorescence without loss of the amplified signal.¹ The cells were trapped in closed microfluidic chambers which ensures that the fluorescent product expelled from the cell is accumulated in small volumes around the cell in these chambers thus stopping the diffusion loss of enzyme amplified fluorescence. The short mixing time of the FDG reagent and the rapid efflux rate at the cellular membrane ensure that the fluorescence signal outside the cells accurately reflects the enzymatic activity happening in the cell. The requisite microfluidic device is made using a soft polymer, polydimethylsiloxane (PDMS), and consist of a flow layer that contains the cells and a top control layer as shown in figure 1. The actuation of the two adjacent valves in the control layer forms an enclosure of the dimensions 100*100*10 µm³ in which the cells are trapped and cultured. The experiment was carried out by mounting the microfluidic chip onto an inverted fluorescence microscope and was translated by a motorized stage, thus allowing multiplexing of the data acquisition by repeated scans on the chamber, carrying out a single scan of 100 chambers in 2 min typically. As shown in figure 1 fluorescence is exited by a tightly focused laser beam that does not directly illuminate the cell, which ensures no cellular auto fluorescence and photo-damage to the cell do not happen.

Figure 1: Schematic diagram of the microfluidic chamber 

used for the enzymatic assay.¹

Initially experiments were carried out to test the system for single molecule measurements. This was done by injecting β-gal enzyme solutions which are very dilute into the chambers and then the fluoregenic fouorescien digalactoside( FDG) substrate was introduced into the chambers. It was observed that the fluorescent signals increase with time, and the slopes of curves shown in the figure 2 give the rates of the hydrolysis.  It was found from these plots that the distribution of hydrolysis rates measured in different chambers showed quantized and evenly spaced peaks which in turn was attributed to the presence of integer numbers of β-gal molecules. Also the spacing between the peaks is 60pMmin^-1., which tells the calibration for the increase in rate of fluoroscein concentration corresponding to one enzyme molecule in the chamber. 

Journals Related to Microfluidics and Nanofluidics

As the field of Microfluidics and Nanofluidics is expanding rapidly, the number of publications related to this field is also increasing. There are some journals that are focused on publishing micro and nano fluidic work and several other journals publish related work. Here is a list of major journals covering microfluidic/nanofluidic, lab on chip, MEMS research.

1. Lab on a Chip (LOC): Publishes on or off chip miniaturization at micro-and nano-scale in chemistry, biology, bio-engineering physics, electronics, clinical/medical science, chemical engineering and material science.

Publisher: Royal Society of Chemistry, UK. 

Impact factor: 6.5 (2009)
Publishing since: 2001; 24 issues per year
Open access: No

2. Microfluidics and Nanofluidics: Publishes all aspects of microfluidics, nanofluidics, and lab-on-a-chip science and technology.

Publisher: Springer 

Impact factor: 2.6 (2013)
Publishing since: 2005; 6 issues per year
Open access: No

3. Biomicrofluidics (BMF): Publishes fundamental physicochemical mechanisms associated with microfluidic and nanofluidic phenomena as well as novel microfluidic and nanofluidic techniques for diagnostic, medical, biological, pharmaceutical, environmental, and chemical applications. 

Publisher: American Institute of Physics 

Impact factor: 3.7 (2013)
Publishing since: 2007; 6 issues per year

Open access: No

4. Electrophoresis: Publishes original manuscripts on all aspects of electrophoresis and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid phase micro-extraction, etc.). 

Publisher: WILEY-VCH Verlag GmbH & Co. KGaA, Germany

Impact factor: 3.1 (2013)
Publishing since: 1980; 24 issues per year

Open access: No

5. Sensors and Actuators B-Chemical: Covers research and development in the field of chemical sensors, actuators and microsystems.

Publisher: ELSEVIER

Impact factor: 3.8 (2013)
Publishing since: 1990; 16 issues per year

Open access: No

6. Analyst: Publishes fundamental discoveries, inventions and applications in the analytical and bioanalytical sciences. It publishes microfluidics and nanofluidics works related to analytical and bioanalytical sciences. 

Publisher: Royal Society of Chemistry, UK
Impact factor: 3.9 (2013)

Publishing since: 1876; 24 issues per year

Open access: No

7. Analytical Chemistry: Devoted to the dissemination of new and original knowledge in all branches of analytical chemistry. It is published by the American Chemical Society and is the most cited journal in this field.

Publisher: American Chemical Society, USA
Impact factor: 5.8 (2013)

Publishing since: 1929; 24 issues per year

Open access: No

8. Computers and fluids: Publish the development of numerical methods relevant to fluid flow computations, computational analysis of flow physics and fluid interactions

Publisher: ELSEVIER
Impact factor: 1.5 (2013)

Publishing since: 1973; 12 issues per year

Open access: No

9. Biosensors and Bioelectronics: Publishes the applications of LOC with integrated sensors, e.g., electrochem., optical sensors. 

Publisher: ELSEVIER
Impact factor: 6.4 (2013)

Publishing since: 1990; 12 issues per year

Open access: No

10. Biomedical Microdevices: Publish research in the diagnostic and therapeutic applications of LOC devices.

Publisher: Springer
Impact factor: 2.7 (2013)

Publishing since: 1998; 6 issues per year

Open access: No

What comes next in Microfluidic research?

Prof George Whitesides sees the future of microfluidics research in following way. This is an editorial published on Lab on a Chip, a leading Journal in this field ((DOI: 10.1039/c0lc90101f)).

Reinvention

Every field must periodically  reinvent it- self to remain vital. Microfluidics, and the concept of the lab-on-a-chip (LoC), have had a spectacularly successful 15-year run of science and technology.  The combina- tion  has  achieved  much  more  than  one could have imagined at the beginning, but also less than one might have hoped for in the most expansive of visions. There are now two strategies—two  paths—for  it to follow in going forward:  (i) It can gather up the technology  that  is now available, and develop it fully and completely. This strategy  would focus on finding uses for what now exists, and motivate  the devel- opment  of  downstream   technologies— manufacturing at  scale, quality  control, standards,   interfaces,   regulatory clear- ance, and all the others—with  those uses. The   development    of   the   downstream manufacturing technologies  will be chal- lenging,  and absolutely  necessary  before laboratory prototypes become large-scale commercial realities. (ii) It can invent new things, and see if the momentum of new ideas  will carry the  field  forward.  This strategy   does   not   necessarily   directly result in products,  but it demonstrates the components   and  options   which  would support later technologies. Sexy new ideas also  build enthusiasm  for  the  field, and demonstrate capabilities that—in an area that  really is new—are  unique, and  that give  rise  to  applications  that   pull  the science into technology.

Both   paths   are   important  for   LoC technology:   It   must,   of   course,   push technology into products  in order to have the impact that will sustain the field. Still, the period of highly productive  invention and  science in LoC  systems  and  micro- fluidic technology  is hardly over, so new ideas are also important.

Everyone active in the field could make up  a  list  of  exciting  opportunities; the length   of  an aggregated   list,  and   the diversity  of  the opportunities  suggested, would provide one measure of its vitality. I would not presume to offer a canonical list,  but  I  will  summarize a  few of  my favorite topics, to give a sense for where I see attractive  opportunities. Let me give you some of my favorites in both strategies: that  is, in invention  of  new science and technology and in the development of existing technology.

1.  Nanofluidics

Microfluidics is the study of fluids moving in   micron-scale   (usually,   to   be   more accurate,  100-micron-or  submillimeter- scale-)    structures.    There    are    many interesting  characteristics   of  these now- familiar  systems,  of  which  perhaps  the most   different    by    comparison   with macroscopic fluid flows, and thus, so far, the  most  useful,  has  turned   out  to  be laminar,  or low Reynolds-number, flow. The field of ‘‘microfluidics’’  has  not  yet made  a  concerted  effort  to  understand true nanofluidics (which I will characterize as  the  behaviors  of  fluids  in  structures with dimensions from 1 to 100 nm). It is not   quite clear  why   microfluidics   has extended  only  slowly  into  nanofluidics, but one reason is probably  that there are still no methods of fabrication that would make   it   easy   to   generate   nanofluidic structures,    or   that   would   allow   the behaviors  of  fluids in them  to  be easily characterized.

‘‘The field of ‘microfluidics’ has not yet   made  a  concerted  effort  to understand true nanofluidics.’’

And   why  would  one  care?  What  is interesting    to    me   about    nanofluidic systems, in principle, is that the behavior of fluids in nanochannels will probably  be dominated    by   their   proximity   to   the surfaces   making   up   the   walls   of  the channels  (or  other  structures contacting the fluids). Nanofluidic systems will be, in other words, ‘‘all interface,’’ and the study of  nanofluidics  may  ultimately  become more a branch  of surface science than an extension   of   microfluidics.   Regardless, one  of  the  least understood, and  most important,  areas   of  science  and   tech- nology is that of interfacial fluids. Storage of  energy  in   capacitors   and   batteries, corrosion,  lubrication, molecular  recognition,    sensing, adhesion,   biocompati- bility—all are intimately concerned  with the properties of fluids (and, of course, of the molecules and ions in them) that  are making the transition from a constrained environment (immediately adjacent to the surface) to the bulk fluid.

2.    Digital microfluidics

The   microfluidics/LoC   community   has begun  actively  to  embrace  the  study  of dispersed    phases    moving    in    micro- channels.  The  invention of a number  of simple  structures  (flow-focusing nozzles, T-junctions,  and others) opened the door to this field by making it possible—for the first   time—to    generate    monodisperse droplets  or bubbles in virtually unlimited numbers, very rapidly (bubble generation rates  now  approach  100  kHz);  micro- fluidic   channels   make   it   possible   to manipulate  and  sort  and  combine  these droplets with remarkable  sophistication. Each  of  these droplets,   in  principle,  is a  micro-reactor,  and  the  potential  for using droplets  for a wide range of appli- cations—genomics,   proteomics,    single- cell                     analysis,       cell           selection, phage selection, many others—seems very large, albeit   still  at   an  early   stage.  ‘‘Digital PCR’’   (from which    phrase    I   adapt ‘‘Digital   Microfluidics’’)   is   developing rapidly, but other uses for these systems— in  biology,  in  food  science,  in  a  wide variety  of  different  types  of  analyses— coupled  with  the  remarkable  self-orga- nizing  properties   of  large  numbers   of droplets  in  microfluidic  systems,  makes this area, to me, extraordinarily attractive for exploratory research.

3.    Inside biology

One of the long-term justifications for the relevance of microfluidics to biology has always been that  it provides information concerning fluid flows in cells and organ- isms. Organisms with a circulatory system are,   essentially,   networks   of  pipes   of various sizes which transport fluid among its   various   parts.   Flows   of  fluids   in biology  range  from  turbulent  in  large pipes (e.g., the aorta),  to laminar in small

ones (e.g., capillaries); these flows can be either                 highly      non-Newtonian       (the contents  of the bowel, the cytosol filling the  interior  of  the  cell)  or  Newtonian (normal  urine);  they  can  have multiple dispersed phases (cells or clots in blood or lymph,  or pathogens  in the  circulation), or   be   (we   think)   homogeneous;    the structures of the channels can range from simple tubes to complex, networked,  gel- filled   capillaries,  and  the immensely complex    networks    of   structure    and architecture     constituting   the    human circulatory  and lymphatic systems (which we   barely   understand). Most serious observations of fluidics in biology  have discovered behaviors  that  are interesting and unexpected.  Since almost  everything in biology  has  layers of complexity  that extend  apparently without   limit,  if  the first  surveys  of  biological  microfluidics have revealed as much of interest as they have, we can  only  begin  to  guess what more serious investigation  will reveal.

‘‘Most of the cards in the microfluidic deck have still not been revealed!’’

4.    New types  of uses

Partially as a reflection of the history of its origin  (and  partly,  probably,  since it  is where the money for start-up companies has  largely  been), developments  in LoC technology  have strongly emphasized bi- oanalysis,  particularly  bioanalysis   rele- vant to human healthcare and to research biomedicine.  This field is enormous  and diverse,   and   these   developments   will certainly continue. The field should think about opportunities in other areas as well. In  applied  biology,  there  are  a  range  of opportunities in plant and animal health, and largely untouched potential for use in public health  (as  opposed  to  high-tech- nology medicine): vaccination  status and nutritional   status    are   two   in   which convenience and  very low cost  are espe- cially important. Fluidic  optics—the  use of fluid-filled  channels  as optical  wave- guides  and  lasers,  and  of  droplets   for lasing, and for uses of lasing such as in- cavity detection—is attracting substantial interest. Systems of droplets or bubbles in complex  microfluidic behaviors indicate massively   parallel   interactions   among them, and suggest the possibility of use in new  kinds  of  analog  computation, and possibly  in  transmission of information.

The use of microfluidic systems in organic synthesis  is no  longer  a  new  idea,  but applications  successful  enough  to  drive the field still remain to be developed. The uses of magnetic separations in all areas of  microanalysis  have  just  begun  to be examined. Combinations of microfluidic systems,  compressed   gases,  and   liquid metals, offer potential  routes to the solu- tions of problems in soft robotics. Most of the  cards  in the  microfluidic  deck  have still not been revealed!

5.    Cheap, interconnectable, stackable systems

One of the surprises of microfluidic systems is that  although the technology has devel- oped well, LoC systems are still not being used  extensively. There are various  argu- ments for what is now required to make the next  step,  but  one  guiding  lesson  from other  fields of  microscience  is ‘‘cheap  is good;’’ the second is ‘‘they should be easy to build  with.’’  Microelectronics   has  pros- pered  because  it learned  (in fact,  taught) both lessons; silicon MEMS, by contrast, has developed much more slowly, in part because  ‘‘cheap’’  has  been  difficult.  And even   in    microfluidics,  polymers  have essentially   displaced   silicon   and   glass, largely because of cost.

Although    the   field  of   microfluidics strives to  draw  analogies  between  LoC systems    and  integrated    circuits,    the analogy   is   fundamentally   weak.   The parallel  fabrication,  and  ease  of  inter- connection, available  with silicon micro- electronics   simply,  at   present,   has   no parallel   in  LoC  technology,   and  even inexpensive polymer systems are dramat- ically  more  expensive than  microcircuits of                comparable         functionality       and complexity. Taking microfluidics to a new plateau of cost and interconnectivity may not  require  fundamentally new  science, but it will require  a change in the objec- tives of the engineering: the rapid devel- opment   of  simple  microfluidic  systems based on patterned paper for applications  in  public  health  in developing  countries provides   an  example  of  the  power  of

‘‘cheap’’ and ‘‘simple.’’

‘‘Taking  microfluidics  to  a  new plateau of cost and interconnectivity may ... require a change in the objectives of the engineering.’’

6.    New fluids, fluidics, and materials

The field of microfluidics has had a very restricted view of fluids and the materials used  to  contain   them,  and  LoC  tech- nology       has                 worn   complementary blinders.  Much  of  the  work  in  micro- fluidic systems is implicitly focused on the objective  of  bioanalytical  systems,  and thus has found it quite satisfactory  to use commercial   polymers,   and   to   assume water   or   an   aqueous   solution   as  the working  fluid. What could be done with fundamentally different  fluids, and what materials might be required to work with them?  As one  example,  many  inorganic materials  (for   example,   glass,  calcium phosphate,   silicon)   form   low-viscosity fluids  at  sufficiently  high  temperatures. What   could  be  done  by  manipulating such  high-temperature  fluids  using  mi- crofluidic  systems?  What  would  be  the behaviors  of  high-temperature melts  of glasses or metals moving through  appro- priate  systems? Could  one  make  micro- electronic systems, or silicon MEMS, by molding   liquid   semiconductors?   Solar cells? LEDs?  Could  one  assemble  more complex systems using techniques related to cofabrication?  What  about  the micro- fluidics of flames and  plasmas?  Most  of these  exploratory  efforts  would  require new methods  of fabricating  microfluidic systems   in   unfamiliar    materials    (e.g. zirconium oxide, thorium oxide, graphite, and niobium have excellent high temper- ature   properties,   but   how   would   one fabricate   microsystems   in  them?  Even more to the point,  how would one char- acterize the movement of fluids in them?). On a more mundane  level, is it incon- ceivable to make microsystems fabricated  in glass as inexpensive as those fabricated  in  polymers?  To  do  so  would  certainly solve many of the problems that  appear when using reactive solvents in polymer-based microfluidic systems.

7.    Interfaces  and standards

The  subject  of interfaces  and  standards might seem boring,  but to a technologist they are  not,  they are  essential  parts  of building any  new  technology.  Think  of what   the   simple   USB   connector has done   for   microelectronics.    Think   (or learn) about  all of the standards that go into  microelectronics,  or components automobiles,    or    open    software,    or household   electrical  systems.   Complex technologies almost      always     have multiple    parts,    and    the   parts    must connect  with  one  another   transparently and  effortlessly,  so that  designers  know that  the  component they  are  designing can   be  connected,   and   so  that   users know  how  to  put  components   together (with  the ssurance  that  they  will work once   connected).   The   field  of  micro- fluidics   is   beginning   to   think   about interfaces and standards, but there is still disagreement  about  whether  the  time  is ripe for a serious effort to design and set standards.   Setting    standards   is,    in a sense, imposing a freeze on design, and one does not want to do it in a way that limits  creativity  and  slows  the  develop- ment  of new  systems. Nonetheless,  until there   are   standards,  it   is   effectively

impossible  to  build  a  technology  of  in- terconnected components, and  the crea- tivity  of  down-stream   designers   –  the designers  whose  skill is to  take  existing components  and  put  them  together   in creative  new ways – is blocked  without understood standards. There  is also  no free  lunch,  and  building  interfaces  and standards requires   work.       It              may, however,  be appropriate,  and  necessary, now.  Thinking   through   standards and interfaces, prototyping systems  demon- strating   them,   and  developing  a  tech- nology  for  them  for  LoC  systems  will

Lack of standards and inter-connectivity poses a potential barrier to  the creativity of designers.

require  great ingenuity,  and  will be  very important for the field.

These seven topics are purely personal choice: any reader could come up with an equally   good   list, and   the   list  would probably  be  quite  different.  That  fact— that there is a wide range of opportunities, and a wide range of opinions  on what is more  important and  what  is  less impor- tant—gives a measure of the health of the field. So long as there are lots of oppor- tunities, and lots of differences in opinion, the  broad area  encompassing  LoC  and microfluidic   technology   (and   plausibly extending into a range of other  subjects, from energy storage and robotics  to low- cost MEMS)  is in good shape.  Disputa-  tion is good.

George M. Whitesides 

Chair, Editorial  Board Harvard University, USA