K Hölz1, J Lietard1, M M Somoza1. 1. Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna , Althanstraße 14 (UZA II), A-1090 Vienna, Austria.
Abstract
Ultraviolet light emitting diodes (UV LEDs) have become widespread in chemical research as highly efficient light sources for photochemistry and photopolymerization. However, in more complex experimental setups requiring highly concentrated light and highly spatially resolved patterning of the light, high-pressure mercury arc lamps are still widely used because they emit intense UV light from a compact arc volume that can be efficiently coupled into optical systems. Advances in the deposition and p-type doping of gallium nitride have recently permitted the manufacture of UV LEDs capable of replacing mercury arc lamps also in these applications. These UV LEDs exceed the spectral radiance of mercury lamps even at the intense I-line at 365 nm. Here we present the successful exchange of a high-pressure mercury arc lamp for a new generation UV LED as a light source in photolithographic chemistry and its use in the fabrication of high-density DNA microarrays. We show that the improved light radiance and efficiency of these LEDs offer substantial practical, economic and ecological advantages, including faster synthesis, lower hardware costs, very long lifetime, an >85-fold reduction in electricity consumption and the elimination of mercury waste and contamination.
Ultraviolet light emitting diodes (UV LEDs) have become widespread in chemical research as highly efficient light sources for photochemistry and photopolymerization. However, in more complex experimental setups requiring highly concentrated light and highly spatially resolved patterning of the light, high-pressure mercury arc lamps are still widely used because they emit intense UV light from a compact arc volume that can be efficiently coupled into optical systems. Advances in the deposition and p-type doping of gallium nitride have recently permitted the manufacture of UV LEDs capable of replacing mercury arc lamps also in these applications. These UV LEDs exceed the spectral radiance of mercury lamps even at the intense I-line at 365 nm. Here we present the successful exchange of a high-pressure mercury arc lamp for a new generation UV LED as a light source in photolithographic chemistry and its use in the fabrication of high-density DNA microarrays. We show that the improved light radiance and efficiency of these LEDs offer substantial practical, economic and ecological advantages, including faster synthesis, lower hardware costs, very long lifetime, an >85-fold reduction in electricity consumption and the elimination of mercury waste and contamination.
Entities:
Keywords:
Biopolymer microarray synthesis; Energy efficiency; Green photochemistry; Spatial patterning; Ultraviolet light emitting diode
Light is a highly versatile
energy source for triggering and controlling
chemical reactions.[1] Unlike conventional
reagents, light can be conveniently generated during use and leaves
behind only the completed photochemical reaction.[2] An important additional benefit is the possibility to use
light to direct reactions with both high temporal and spatial accuracy.
Spatial patterning of light can be obtained using an optical imaging
system and is the basis for photolithography. Photolithography, usually
by means of light-directed polymer chemistry, includes not only the
synthesis of integrated electronic circuits and most printed materials
but also many more specialized chemical applications, such as three-dimensional
fabrication and printing,[3,4] surface functionalization,[5] and the synthesis of biopolymer microarrays.[6−12] The primary drawback to photochemistry has been the inability of
most molecules to absorb visible light efficiently, requiring specialized
sources to generate the necessary ultraviolet radiation. This concern
has driven significant interest in solar and visible light photochemistry,
as a more continent and sustainable source of light for organic transformations.[13−16] Daylight itself, however, is complex to control accurately and reliably
in most industrial or research settings. Artificial sources of white
light are very inefficient, particularly because, in almost all cases,
only certain wavelength ranges are relevant. Furthermore, because
most sources, such as low pressure mercury, incandescent, and excimer
lamps, emit from extended surfaces or volumes, the light cannot be
efficiently collected and directed to a reaction site. Recent advances
in the manufacture of ultraviolet light emitting diodes (UV LEDs)
allow very energy efficient, reliable, and simple generation of the
near UV light, which is most suitable for photochemistry and chemical
photolithography. These LEDs are simple to assemble into useful light
sources capable of outperforming traditional sources of high intensity
UV light, particularly mercury arc lamps, which are widely used in
photochemistry applications. Mercury vapor lamps are quite efficient
in converting electricity to light and have found widespread use.
For many scientific and technical applications, the ultraviolet component
of high-pressure mercury lamps makes them particularly useful because
few other light sources efficiently generate such large amounts of
UV light. Another major advantage of high pressure mercury arc lamps
is that the light is generated in the small arc volume between anode
and cathode. The small source size is important in any application
where an optical system is used to transfer the light to a target.
The optical principle of geometrical extent, or étendue, dictates that efficient light power transmission requires a small
source.[17] Because of these two key advantages,
efficient UV generation and small source size, high pressure mercury
arc sources have been essential tools in all areas of photochemistry
and chemical photolithography for many decades. Nevertheless, these
light sources have a significant drawback. Mercury is acutely toxic
and is a major persistent environmental pollutant.[18,19] The high pressure arc lamps of the sizes typically used in laboratories
contain hundreds of milligrams of mercury, necessitating careful storage,
handling and disposal.[20] The lamps also
have short lifetimes, usually in the range of 500 to 1000 h, requiring
frequent and costly replacement. The short lifetime is exacerbated
by their inability to withstand multiple on–off cycles, so
that in many applications they must be operated continuously throughout
their lifetimes.[20] Also, because many applications
make use of a single spectral line, often the very intense I-line
at 365 nm, the majority of the light output must be filtered out,
resulting in very poor overall energy efficiency.Recent advances
in the manufacture of gallium nitride-based LEDs
with indium gallium nitride (InGa1–)N quantum wells have provided very
efficient solid-state sources of blue light.[21] These sources, in conjunction with phosphors to generate additional
colors, are rapidly replacing incandescent and low pressure fluorescent
mercury lamps in general ambient lighting.[22,23] The same technology can be used to generate near UV light very efficiently
(external quantum efficiency: ∼50%) at the same wavelength
as the very strong mercury I-line at 365 nm.[24,25] In addition to high quantum efficiency, these LEDs have very long
lifetimes and can be switched on and off as needed, which further
enhances their overall energy efficiency and practical lifetime.Only in the past year have surface-mount UV LEDs that can compete
very favorably with mercury arc sources become available. Here we
describe a simple high power 365 nm UV LED source that significantly
outperforms mercury arc sources in applications in photochemistry
and chemical photolithography. Specifically, we have constructed and
evaluated such an LED source for maskless array synthesis (MAS), a
chemical photolithographic application that uses an optical imaging
system centered around a digital micromirror device (DMD) to direct
the chemical synthesis of complex microarrays of biomolecules, such
as nucleic acids and peptides, via the selective removal of photolabile
protecting groups.[8−10,26−28] In this type of imaging system, the complexity of the optics and
the requirements for a very small numerical aperture (NA) result in
very low light throughput.[29] When a 350
W ultrahigh-pressure Hg arc source is used, which generates ∼20
W of 365 nm photons,[30] the system transmits
to the target only about ∼0.6% of the total lamp emissions
of this spectral line. In the case of our UV LED source (Figure ), the LED generates
significantly less light, ∼4 W of 365 nm photons, but because
of the small size and optimal geometry of the emitting surface, the
absolute amount of light reaching the target is higher, about 140
mW, or ∼3% of the output. The 5-fold increase in throughput
contributes to an >85-fold overall energy efficiency increase.
Additional
advantages, such as greatly increased expected lifetime, simplicity,
reliability and low cost, are discussed in the Results
and Discussion section.
Figure 1
Exploded and assembled view of the ultraviolet
light source. The
UV source, a Nichia NVSU333A U365 LED is soldered directly to a 6
mm thick solid copper block to which copper anode and cathode terminals
have been affixed with electrical isolating, high-temperature epoxy.
The LED die heat-sink terminal is soldered directly to the copper
block, providing a very efficient thermal path to the CPU cooler heat
sink and fan.
Exploded and assembled view of the ultraviolet
light source. The
UV source, a Nichia NVSU333A U365 LED is soldered directly to a 6
mm thick solid copper block to which copper anode and cathode terminals
have been affixed with electrical isolating, high-temperature epoxy.
The LED die heat-sink terminal is soldered directly to the copper
block, providing a very efficient thermal path to the CPU cooler heat
sink and fan.
Experimental
Section
UV LED Source Construction
The source consists primarily
of a single Nichia NVSU333A U365 surface-mount LED,[31] a heat-spreading copper block with embedded anode and cathode
terminals, a CPU heat sink and fan, and an aspheric condenser lens
(Figure ). Only LEDs
from the P15d21 radiant flux ranks (3760 to 4100 mW) were available;
the Nichia manufacturing process also yields 365 nm LEDs in radiant
flux ranks up to P16d22 (4870 to 5310 mW), but the higher power rank
LEDs could not be obtained. The LEDs have a rank-dependent, typical
conversion efficiency of ∼27% when operated with a forward
current of 3.5 A and well cooled. At a forward voltage of 3.85 V,
this corresponds to a thermal dissipation of 11 W. Because the mass
of the LED is only ∼0.25 g, a very efficient thermal dissipative
path is required, particularly because both the radiant flux and lifespan
of LEDs are inversely related to the temperature of the emitting semiconductor
die. A 40 × 40 × 6 mm copper block was used to transfer
heat away from the LED. The copper block includes two slots fitted
with copper anode and cathode terminals. Each terminal was electrically
isolated from the block with high-temperature epoxy (Aremco-Bond 526-N).
After epoxy curing, the front face of the copper block was sanded
to a smooth and flat finish in preparation for soldering.The
back sides of the LEDs have a large central die heat-sink pad flanked
by the anode and cathode pads. Each of these was soldered to the copper
block and terminals, respectively, using the recommended soldering
pad pattern. The soldering pad pattern was cut into ∼50 μm
thick packing tape, which was then adhered to the copper block and
terminals. The solder paste (EDSYN SR11 Sn42-Bi58 low-temperature
solder alloy) was spread over the tape solder stencil using a razor
blade. After the stencil was peeled off, the three LED pads were carefully
aligned with the appropriate solder pads. The copper block was then
placed on a laboratory heat plate, set to 180 °C, until the solder
melted (138 °C). After it was checked that the heat sink and
terminals remained electrically isolated from each other, the copper
block was removed from the heat plate to cool slowly to room temperature.
The copper block was then thermally coupled to a CPU heat sink (Arctic
Copper Silent 3 AMDCPU cooler) using a thin layer of thermal grease
(Shin-Etsu X-23-7921-5), and then fastened with four screws. Light
from the emitting surface was focused using a 10 mm diameter 6.6 mm
FL aspheric condenser lens (Edmund Optics #88-283). Four screws on
the lens holder allow accurate positioning of the lens relative to
the LED.
LED Power Supply and Control
Because current flow through
LEDs increases exponentially with voltage, constant-current supplies
are used to accurately control light output. In addition to a power
supply, a trigger circuit is necessary to turn the current on or off
under computer control. Figure A shows a schematic for the LED control electronics. An adjustable
power supply in constant current mode (B&K Precision 1687B) provides
an adjustable regulated current for the LED. Computer switching control
is enabled by a low on-resistance n-type power MOSFET
(IR AUIRFB8409) in parallel with the UV LED. A 5 V trigger signal,
electrically isolated from the power circuit via an optocoupler, disables
current flow through the MOSFET, turning on the LED. In the absence
of the trigger signal, resistance between the MOSFET drain and source
terminals is close to zero, forcing the constant-current source to
reduce its output voltage to close to zero and hence eliminating any
current flow through the LED, which has a conduction threshold of
∼3 V. Interrupting the current through the LED with a shunt
circuit is advantageous because it forces the constant-current supply
to reduce its output voltage, which in turn prevents damage to the
LED from a current spike each time the LED is turned back on. The
electrical characteristics of the circuits were measured using a Rigol
DS1054 oscilloscope. The power supply output response is shown in Figure B,C. With this circuit,
the time to reach full power depends on the response time of the current
source; in this case about 30 ms, whereas the turn-off time is close
to instantaneous due to the fast (∼30 ns) response time of
the MOSFET.
Figure 2
(A) Power and control circuit for the Nichia NVSU333A U365 LED.
An adjustable current source powers the LED. The LED is turned on
or off via an n-type MOSFET shunt triggered by a
5 V logic signal. Oscilloscope traces for trigger on (B) and trigger
off (C) (blue) show that the circuit behavior is optimal, with a 30
ms soft-start output voltage to the LED (red) and an instant turn
off (10 ms per div. horizontal and 50 and 800 mV per div. vertical
for blue and red channels, respectively).
(A) Power and control circuit for the Nichia NVSU333A U365 LED.
An adjustable current source powers the LED. The LED is turned on
or off via an n-type MOSFET shunt triggered by a
5 V logic signal. Oscilloscope traces for trigger on (B) and trigger
off (C) (blue) show that the circuit behavior is optimal, with a 30
ms soft-start output voltage to the LED (red) and an instant turn
off (10 ms per div. horizontal and 50 and 800 mV per div. vertical
for blue and red channels, respectively).
Intensity and Stability Measurements
LED intensity
stability was measured using (1) passive cooling (heat sink fan off),
(2) fan cooling (heat sink fan on), and (3) regulated active cooling
using a Peltier thermoelectric device between the copper heat sink
and the CPU cooler. For the thermoelectric cooling, the hot and cold
sides of a Peltier device (European Thermodynamics APH-161-12-14-E)
were thermally coupled to the corresponding parts using thermal paste.
The Peltier device was regulated at set points of either 16 or 26
°C using a Willhi WH7016C thermostat and a temperature sensor
in thermal contact with the copper heat sink immediately adjacent
to the thermal pad of the LED. To maximize the thermal contact, a
dimple matching the curvature of the sensor was drilled into the copper
and thermal paste was used between the two surfaces. The relative
intensity was measured using a photodiode (Vishay BPW21R) and logged
using a UNI-T UT61B multimeter connected to a computer.
Microarray
Synthesis and Hybridization
Microarrays
were synthesized using a chemical photolithographic method termed
Maskless Array Synthesis (MAS).[10,29] MAS uses a computer
controlled optical imaging system, synchronized with a chemical delivery
system, to direct the stepwise synthesis of complex, high-density
microarrays of nucleic acid oligomers on glass substrates. A UV light
source and associated condenser lens(es) fill the entrance to a light
pipe, which spatially shapes and homogenizes the intensity of the
light beam via multiple reflections on the first surface aluminum
mirrors that constitute the inner surface of the light pipe.[32] The output of the light homogenizer functions
as a rectangular, highly spatially homogeneous source for illuminating,
via a 2× magnification telescope, a 0.7″ (diagonal) digital
micromirror device (DMD). The DMD is imaged, via 1:1 Offner relay
optics, to the surface of the substrate in the reaction chamber[33] where the microarray synthesis is taking place.
One of two tilt positions of each of the mirrors of the DMD, either
ON or OFF, determines whether photodeprotection will take place at
each microarray position. The low numerical aperture of the system,
combined with the large number of reflections needed to form a highly
homogeneous beam of light, as well as to modulate it spatially and
direct it to the target, results in very low system light throughput.
The optimum source has previously been a 350 W Hg arc lamp, which
has an effective plasma size (0.8 × 2.7 mm) that is a reasonably
good match to the entrance optics. Higher power lamps have larger
plasma dimensions which would overfill the entrance pupil and not
result in the collection of significantly more light. The mercurylamp source is a 350 W high pressure short arc mercurylamp (Newport
6286) in an F/0.7 500 W housing (Newport 66905). The light from the
lamp was filtered using two 350–450 nm primary-range dichroic
mirrors (Newport). Ozone generated in the lamp housing was catalytically
converted to O2 using an Ozone Eater (Newport 66087). For
both the mercurylamp and the UV LED, the radiant intensity of light
reaching the reaction cell was measured using a calibrated SÜSS
MicroTec Model 1000 UV intensity meter with a 365 nm probe.Microarray synthesis chemistry is quite similar to the standard phosphoramidite
chemistry used in solid phase synthesis.[34] The primary modification is the use of a (2-(2-nitrophenyl)propyloxycarbonyl
(NPPOC) photolabile protecting group in the place of the acid labile
dimethoxytrityl (DMT) group. During photodeprotection, the reaction
chamber is filled with a solution of 1% imidazole in DMSO, as the
photoinduced β-elimination pathway of the NPPOC group is favored
by the presence of a small amount of an amine base.[35] The substitution of light for the acid deblocking step
greatly reduces the requirement for oxidizing the phosphite triester
resulting from the coupling reaction, which is unstable under acidic
conditions, to the fully stable phosphotriester.[34] Thus, photolithographic microarray synthesis, in comparison
with normal solid-phase synthesis, eliminates the health and environmental
toxicity associated with the use of the deblock solution (di- or trichloroacetic
acid in dichloromethane), and greatly reduces the use of tetrahydrofuran,
as well as pyridine, a “red category” solvent that is
an essential component of the oxidizer solution.To test the
light exposure needed to remove the NPPOC or SPh-NPPOC
5′-hydroxyl protecting groups on the growing oligonucleotide,
and to verify that the exposure and synthesis outcome using the UV
LED source are the same as with a Hg source, microarrays bearing a
single oligonucleotide sequence (5′-GTC ATC ATC ATG AAC CAC
CCT GGT C) were synthesized, but photodeprotection was carried out
using a spatial gradient of light exposures.[29,36] After synthesis, the protecting groups on the DNA oligonucleotides
were removed using a solution of 1:1 (v/v) ethylenediamine/ethanol
for 2 h at room temperature. After the deprotection solution was washed
off with deionized water, the exposure gradient microarrays were hybridized
for 2 h at 42 °C with the Cy3-labeled complementary sequence
and then washed. The hybridization buffer consisted of 150 μL
of 2× 2-(N-morpholino)ethanesulfonic acid (MES),
110 μL of nuclease free water, 13.3 μL of acetylated bovineserum albumin (BSA), and 26.7 μL of 100 nM 5′-Cy3-labeled
complementary oligonucleotide. The washing steps start with vigorous
shaking in a 50 mL centrifuge tube filled with 30 mL nonstringent
wash buffer (SSPE; 0.9 M NaCl, 0.06 M phosphate, 6 mM EDTA, 0.01%
Tween 20) for 2 min, followed by a stringent wash (100 mM MES, 0.1
M NaCl, 0.01% Tween 20) for 1 min, and a quick dip in final wash buffer
(0.1× saline–sodium citrate buffer) to remove excess salt.
The arrays were imaged in a microarray scanner at a resolution of
5 μm using the 532 nm laser and the 550–600 nm bandpass
filter. Data was extracted from the scan image using NimbleScan v2.1.
Data was normalized and fit with the four parameter sigmoidal function
in Sigmaplot.
RESULTS and DISCUSSION
UV LEDs with
useful outputs have been available for several years
and have been used in photolithography[37,38] and photochemistry,
primarily as arrays of LEDs, to expose and cure inks, adhesives, and
coatings. In chemistry research, they have been used in recent years
primarily for photopolymerization and photocatalysis.[39−43] However, in all of these applications the UV-LED output was directed
onto the reaction site without the use of an optical system. For chemical
photolithography, such as surface functionalization patterning and
microarrays synthesis, the optical requirements are often very strict
in order to achieve spatial control with lateral resolution of a few
micrometers over surface areas of a few square centimeters. For these
applications, a very large percentage of the emitted light cannot
be coupled into the optical system and/or is lost in the optical system.
We have found that with the current generation of 365 nm UV-LEDs,
these losses are significantly lower than with mercury arc sources,
which, combined with the very high radiance from the LED, results
in improved light throughput, far lower electricity and resource consumption.
UV LED
Output, Stability, and Lifetime
The 365 nm output
of the Nichia NVSU333A U365 surface mount LED is significantly lower
than that of a high-pressure Hg lamp, ∼4 vs ∼20 W. Nevertheless,
in most applications in photochemistry and chemical photolithography,
the relevant metric is the amount of light that can be collected and
directed to the reaction chamber where the chemistry is being performed.
This metric greatly favors the LED because the light emission from
the LED surface follows a Lambertian pattern, I(θ)
= I0cos(θ), where I0 is the intensity normal to the surface, and because
the emitting surface is small and geometrically simple. This is a
distinct advantage over arc sources, which emit almost isotropically
and from an irregularly shaped plasma, and therefore make light collection
complex and inefficient. In addition, the size of the quartz envelope
and the high heat output of the plasma require much larger lenses
placed more distally in the case of arc sources. Efficient LED light
collection can make use of either reflective or refractive optics
or a combination of both, and may be nonimaging.[44,45] In the case of the Nichia NVSU333A LEDs, and our application in
chemical photolithography, a single 10 mm diameter, 6.6 mm FL aspheric
condenser lens appears to be an inexpensive, near optimal solution.
The small focal length and high NA of aspheric condenser lenses allows
them to be placed a few millimeters from the emitting surface in order
to collect and collimate more light. With a back focal length of 3.3
mm, approximately 75% of the light emitted by the LED is collected
by the lens. Because the LED is not a point source, but has an emitting
surface with dimensions of 2 × 2 mm, the light is not fully collimated
by the lens; instead, the light exiting the lens has an angular divergence
of about 5°, similar to the light acceptance angle determined
by the 0.08 NA of the imaging optics of our photolithographic system.
Such a low NA is necessary to reach a sufficient depth-of-focus (DOF
∝ λ/NA2; ∼60 μm), to achieve
a sufficient lateral resolution (r ∝ λ/2NA;
∼2.7 μm) and to reduce synthesis errors due to scattered
light, which scales ∝ NA2.[46] In this context, more than 140 mW of 365 nm light (100 mW/cm2 over an area of 1.4 cm2), about 3% of the output
of UV LED source, could be transmitted to the target at a forward
current of 3.9 A. This amount is greater than the typical amount of
365 nm light available from the arc lamp in the same imaging system.
The LEDs are rated for forward currents up to 4.5 A, so a small amount
of additional output is available, which may eventually be used to
compensate for LED aging.Sensitive photochemistry experiments,
as well as chemical photolithography synthesis, require light sources
with stable outputs over many hours. Mercury arc sources are fairly
satisfactory in this regard, although the light output decreases steadily
as the plasma erodes the anode and cathode. The erosion degrades the
efficiency and stability of the plasma and deposits tungsten on the
quartz envelope. In our experience and usage pattern with short arc
Hg lamps, the light output drops by ∼1% per day. LEDs have
far greater expected lifetimes, particularly because the LEDs can
be turned on and off as needed. However, because their output is temperature
sensitive and they have very low thermal mass, their output can fall
quickly if they are inadequately cooled. To test the effectiveness
of our cooling system (Figure ), we measured the output intensity for three different cooling
methods: (1) passive cooling (fan off), (2) fan on, and (3) regulated
active cooling using a Peltier thermoelectric device between the copper
heat sink and the CPU cooler.To simulate our normal use, the
LED was run with a current of 3.9
A and was switched on and off at 1 min intervals. The results are
shown in Figure .
With passive cooling, there is a ∼2% drop in output during
each on cycle as well as a steady downward drift in the output due
to a gradual warming of the heat sink. With active fan cooling, the
output drops by about 1% during each on cycle and the longer-term
drift disappears. With Peltier cooling, a somewhat higher light output
can be achieved without output drop. Oscillations in the output during
the Peltier experiments are due to unstable temperature control and
could be minimized with a more complex control feedback loop, such
as proportional-integral-derivative (PID) controller. Because our
application is not sensitive to the 1% drop in intensity during on
cycles, we considered the fan cooling an optimum compromise between
stability and the additional complexity inherent in the thermoelectric
cooling.
Figure 3
Power drifts due to self-heating with and without active cooling.
LED output drops during use at full power (IF = 3.9 A) was monitored for four types of cooling: passive
cooling (fan disconnected), fan cooling, and cooling using a Peltier
element between the copper block and the heat sink. The Peltier cooling
to 16 or 26 °C was thermostatically controlled to ±2 °C.
Power drifts due to self-heating with and without active cooling.
LED output drops during use at full power (IF = 3.9 A) was monitored for four types of cooling: passive
cooling (fan disconnected), fan cooling, and cooling using a Peltier
element between the copper block and the heat sink. The Peltier cooling
to 16 or 26 °C was thermostatically controlled to ±2 °C.
Photolithographic Microarray
Synthesis
To verify that
the chemical photodeprotection afforded by the UV LED is equivalent
to that provided by the mercury arc source, we synthesized microarrays
using a spatial light exposure gradient on the synthesis surface.
With this microarray design, which we use to calibrate optimum light
exposure, e.g., when testing new photolabile group chemistry, only
a single DNA sequence (GTC ATC ATC ATG AAC CAC CCT GGT C) is synthesized
everywhere on the surface, but using a range of exposures from 0 to
10 J/cm2. At low light exposure, microarray oligonucleotides
have a large number of deletion errors and hybridize poorly with fluorescently
labeled complementary oligonucleotides. As the exposure increases,
the number of deletion errors drops until an optimum radiant exposure
is reached. Beyond this optimum, the synthesis time increases due
to the longer exposures but the hybridization signal intensity does
not increase significantly, and in the case of highly complex microarrays
(e.g., gene expression microarrays), synthesis errors increase due
to the increase in scattered light. For microarray synthesis with
phosphoramidites bearing the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC)
photolabile group, we consider 6 J/cm2 as the optimum for
typical hybridization-based DNA microarray applications. For synthesis
with thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPh-NPPOC), the
optimum is 12 times less, 0.5 J/cm2.[36] The results for the comparison between the two light sources
are shown in Figure and demonstrate that there are no synthesis differences between
the sources.
Figure 4
Hybridization intensities for microarrays of a DNA 25-mer
synthesized
with a 356 nm UV-LED exposure gradient using NPPOC (black circle)
or SPh-NPPOC (red triangles) DNA phosphoramidites. Radiant exposure
values for SPh-NPPOC are multiplied by 12.0, respectively, with original
data position indicated by the red dashed line. The solid blue line
is a fit for the equivalent data obtained using the 350 to 450 nm
output of an Hg arc lamp.[36]
Hybridization intensities for microarrays of a DNA 25-mer
synthesized
with a 356 nm UV-LED exposure gradient using NPPOC (black circle)
or SPh-NPPOC (red triangles) DNA phosphoramidites. Radiant exposure
values for SPh-NPPOC are multiplied by 12.0, respectively, with original
data position indicated by the red dashed line. The solid blue line
is a fit for the equivalent data obtained using the 350 to 450 nm
output of an Hg arc lamp.[36]
Lifetime and Electricity Consumption
LED lifetime was
estimated based on 7 months of daily use in the photolithographic
synthesis of microarrays. During this time, 140 microarray syntheses
were performed, with typical cumulative UV exposures of ∼100
min per microarray using an LED current of 3.8 to 3.9 A. Syntheses
with NPPOC phosphoramidites require a photodeprotection radiant exposure
of ∼6 J/cm2, which is achieved in 1 min with a radiant
power of 100 mW/cm2. This power could be achieved using
the LED source, even after 7 months of use, with an LED current of
3.8 to 3.9 A. Photodeprotection in microarray synthesis was performed
by switching on the LED only during the 1 min exposure. The remaining
chemical steps in each synthesis cycle require approximately one additional
minute, during which the LED and heat sink can cool down. This on–off
cycle length was used to estimate the electricity consumption of the
LED system. For this purpose, the electrical power consumption of
the system (LED, power supply and cooling fan) was measured, using
an Arendo power meter, in both the LED on (40 W) and LED off (20 W)
states and consumption was calculated based on typical use patterns,
including turning the system fully off between synthesis. Similar
measurements were used to calculate the equivalent consumption using
a 350 W Hg arc lamp. In this case, the calculations were based on
running the lamp at 380 W during the entire synthesis time (system
power consumption = 480 W) and at an “idle” lamp power
of 200 W between syntheses (system power consumption = 270 W). The
electrical consumption included the arc lamp power supply, lamp cooling
fan, and the fan of the ozone eater. Room cooling was not included
in the calculations but could be expected to add ∼20% to electricity
use.The nominal lifetime of the Newport Hg lamp is 1000 h.
In our usage pattern, in which the lamp is run at an average power
of 380 W during synthesis and 200 W at all other times, we have achieved
an average life of ∼1400 h. Our end-of-life criterion was that
the lamp was unable to supply more than 60 mW/cm2 (0.084
W total power) of 365 nm light to the synthesis plane at an operating
power of 399 W. An attempt to extend effective Hg lamp life by turning
it off between syntheses resulted in a high probability of lamp explosion,
which resulted in significant damage to the optics in the lamp housing
in addition to mercury contamination. The lifetime of the LED source
is too long to be accurately measurable at this point. Nichia estimates
a 10% output drop after 2000 h, even when the LEDs are operated with
a current of 4.5 A, and a lifetime (30% output drop) of 15 000
h when operated at a junction temperature of 70 °C.[47] With our fan-cooled heat sink, the estimated
junction temperature is under 70 °C at an operating current of
4 A; with the Peltier cooling, the junction temperature could be lowered
to under 40 °C at 4 A, corresponding to a theoretical lifetime
of >20 000 h.[47]
Consumption
and Cost
Both the initial investment and
the running costs are important considerations in equipment choice.
Consumable costs are based on the running schedule above and include
only the Hg arc lamp itself or the UV LED. The initial costs to purchase
the light sources and necessary associated power supplies and components
are based on the 2016 prices of the equipment described herein (sources,
housing, power supplies, optics) and are summarized in Table . In the case of the UV LED,
a two year lifetime is assumed primarily because improvements in the
manufacturing process may favor replacing the current UV LED with
a more efficient version. Nevertheless, if the lifetime estimates
by Nichia[47] are correct, our usage pattern
would result in a lifetime approaching 40 years or 10 000 microarrays.
Table 1
Electricity Consumption, Initial Cost
and Consumables Cost Comparison between Mercury Arc and LED Sources
source
electricitya (kWh/year)
initial costb (€)
consumablesc (€/year)
Hg arc
2600
12000
2400
UV LED
30
1500
50
Assumptions: arc
lamp at full power
(380 W) for 20 h/week and at idle power (200 W) 148 h/week; LED at
3.8 A (50% duty cycle for 20 h/week) and fully off 148 h/week.
Estimated 2016 hardware costs.
Six replacement arc lamps/year
or
one LED every second year.
Assumptions: arc
lamp at full power
(380 W) for 20 h/week and at idle power (200 W) 148 h/week; LED at
3.8 A (50% duty cycle for 20 h/week) and fully off 148 h/week.Estimated 2016 hardware costs.Six replacement arc lamps/year
or
one LED every second year.
Conclusions
Ultraviolet LED sources, not only in the near UV region at 365
nm but also up to 400 nm and in the blue and violet regions of the
visible spectrum, are now superior to high pressure mercury arc lamps
in terms of their ability to deliver highly concentrated light via
optical imaging systems. For photochemical applications, UV LEDs now
offer higher near-UV intensities at the reaction site as well as far
lower setup and operating costs while avoiding the use of mercury
and greatly reducing electricity consumption.
Authors: Björn Forsström; Barbara Bisławska Axnäs; Klaus-Peter Stengele; Jochen Bühler; Thomas J Albert; Todd A Richmond; Francis Jingxin Hu; Peter Nilsson; Elton P Hudson; Johan Rockberg; Mathias Uhlen Journal: Mol Cell Proteomics Date: 2014-04-04 Impact factor: 5.911
Authors: Jory Lietard; Hala Abou Assi; Irene Gómez-Pinto; Carlos González; Mark M Somoza; Masad J Damha Journal: Nucleic Acids Res Date: 2017-02-28 Impact factor: 16.971
Authors: Kaiser Pärnamets; Tamas Pardy; Ants Koel; Toomas Rang; Ott Scheler; Yannick Le Moullec; Fariha Afrin Journal: Micromachines (Basel) Date: 2021-03-23 Impact factor: 2.891