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1.
L.L. Holladay 《Journal of The Franklin Institute》1929,207(2):193-230
With the growth of the science of light and vision, the engineer has been putting forth some effort to use daylight to a greater advantage. In our endeavor to use natural and artifical light in a more economical manner, it becomes necessary to know the initial and operating costs of each. The determination of the initial cost of natural lighting is made by computing the difference in cost between a building equipped with natural and artifical lighting and a building of similar construction equipped merely with artificial lighting.Though it may be possible to dispense with natural lighting, it would hardly be possible to dispense with artificial lighting if work is to be pursued for eight hours a day throughout the year; for natural light is of no greater constancy than daylight itself.The major items entering into the initial cost of natural lighting equipment per 100 sq. ft. of floor area under normal conditions vary from a minimum in one type of standard building to a maximum in another type as follows:
5.
Minimum | Maximum | ||||
Windows and structure | $ 0.00 | to | $45.00 | ||
Heating equipment | 10.30 | to | 32.10 | ||
Light-courts | 8.40 | to | 45.30 | ||
Total for all three items for standard buildings | 28.56 | to | 79.90 |
Mol. per cent. AgCl. | Mol. per cent. AgBr. | Lattice spacing 100 Planes. |
0 | 100 | 2.884 |
20 | 80 | 2.862 |
40 | 60 | 2.840 |
50 | 50 | 2.827 |
60 | 40 | 2.815 |
80 | 20 | 2.794 |
100 | 0 | 2.770 |
Sample | Average scores | Standard deviation |
---|---|---|
1 | 33 790 | 368 |
2 | 23 269 | 271 |
3 | 39 598 | 307 |
4 | 7784 | 52 |
5 | 21 222 | 197 |
6 | 38 878 | 290 |
7 | 22 437 | 227 |
8 | 36 295 | 334 |
9 | 41 024 | 396 |
Negative | 200 | 32 |
4.
Keith 《Endeavour》2001,25(4)
In 1971, on the death of Louis Pasteur's grandson, Pasteur Vallery-Radot, the collection of Pasteur's personal papers and notebooks, which had mostly been donated to the Bibliothèque nationale in Paris, became more accessible to scholars. Louis Pasteur (Fig. 1) was one of the world's greatest scientists, but since his death in 1895 his memory has been revered to an extent that almost borders on idolism. One consequence of the improved access to Pasteur's notebooks and correspondence was the publication in 1995 of Gerald Geison's book The Private Science of Louis Pasteur[1], in which Geison compares what he believes to have been the more realistic sequence of steps by which Pasteur reached his unquestionably famous discoveries with the widely publicized Pasteurian legends that often read more like film scenarios. This article attempts to trace the stages by which Pasteur came to some of his celebrated conclusions in the earlier years of his career.
Fig. 1. Pasteur in 1857, aged 34, when Dean of the Faculty of Sciences in Lille (reproduced, with permission, from [1]). 相似文献
Full-size image (18K) |
5.
Rui Zhang Jie Huang Fei Xie Baojun Wang Ming Chu Yuedan Wang Haichao Li Wei Wang Haixia Zhang Wengang Wu Zhihong Li 《Biomicrofluidics》2014,8(3)
Nowadays, microfluidics is attracting more and more attentions in the biological society and has
provided powerful solutions for various applications. This paper reported a microfluidic strategy
for aqueous sample sterilization. A well-designed small microchannel with a high hydrodynamic
resistance was used to function as an in-chip pressure regulator. The pressure in the upstream
microchannel was thereby elevated which made it possible to maintain a boiling-free high temperature
environment for aqueous sample sterilization. A 120 °C temperature along with a pressure of 400 kPa
was successfully achieved inside the chip to sterilize aqueous samples with E. coli
and Staphylococcus aureus inside. This technique will find wide applications in
portable cell culturing, microsurgery in wild fields, and other related micro total analysis
systems.Microfluidics, which confines fluid flow at microscale, attracts more and more attentions in the
biological society.1–4 By scaling the flow
domain down to microliter level, microfluidics shows attractive merits of low sample consumption,
precise biological objective manipulation, and fast momentum/energy transportation. For example,
various cell operations, such as culturing5–7
and sorting,8–10 have already been
demonstrated with microfluidic approaches. In most biological applications, sterilization is a key
sample pre-treatment step to avoid contamination. However, as far as the author knew, this important
pre-treatment operation is generally achieved in an off-chip way, by using high temperature and high
pressure autoclave. Actually, microfluidics has already been utilized to develop new solution for
high pressure/temperature reactions. The required high pressure/temperature condition was generated
either by combining off-chip back pressure regulator and hot-oil bath,11,12 or by integrating pressure regulator, heater, and temperature
sensor into a single chip.13 This work presented a
microfluidic sterilization strategy by implementing the previously developed continuous flowing high
pressure/temperature microfluidic reactor.Figure Figure11 shows the working principle of the present
microfluidic sterilization chip. The chip consists of three zones: sample loading (a microchannel
with length of 270 mm and width of 40 μm), sterilization (length of 216 mm and
width of 100 μm), and pressure regulating (length of 42 mm and width of
5 μm). Three functional zones were separated by two thermal isolation trenches. The
sample was injected into the chip by a syringe pump and experienced two-step filtrations (feature
sizes of 20 μm and 5 μm, not shown in Figure Figure1)1) at the entrance to avoid the channel clog. All channels had the same depth of
40 μm. According to the Hagen–Poiseuille relationship,15 the pressure regulating channel had a large flow resistance (around
1.09 × 1017 Pa·s/m3, see supplementary S1 for details16) because of its small width, thereby generated a high working
pressure in the upstream sterilization channel under a given flow rate. The boiling point of the
solution will then be raised up by the elevated pressure in the sterilization zone followed by the
Antoine equation.16 By integrating
heater/temperature sensors in the pressurized zone, a high temperature environment with temperature
higher than 100 °C can thereby be realized for aqueous sample sterilization. The sample was
collected from the outlet and cultured at 37 °C for 12 h. Bacterial colony was counted to evaluate
the sterilization performance.Open in a separate windowFIG. 1.Working principle of the present microfluidic sterilization. Only microfluidic channel, heater,
and temperature sensor were schematically shown. The varied colour of the microchannel represents
the pressure and that of the halation stands for the temperature.Fabrication of this chip has been introduced elsewhere.14 The fabricated chip and the experimental system are shown in Figure Figure2.2. There were two inlets of the chip. While, in the experiment, only
one inlet used and connected to the syringe pump. The backup one was blocked manually. The sample
load zone was arranged in between of the sterilization zone and the pressure regulating zone based
on thermal management consideration. A temperature control system (heater/temperature sensor, power
source, and multi-meter) was setup to provide the required high temperature. The heater and the
temperature sensor were microfabricated Pt resistors. The temperature coefficient of resistance
(TCR) was measured as 0.00152 K−1.Open in a separate windowFIG. 2.The fabricated chip and the experimental system. (a) Two chips with a penny for comparison. The
left chip was viewed from the heater/temperature sensor side, while the right one was observed from
the microchannel side (through a glass substrate). (b) The experimental system.Thermal isolation performance of the present chip before packaging with inlet/outlet was shown in
Figure Figure3,3, to show the thermal interference issue. The results
indicated that when the sterilization zone was heated up to 140 °C, the pressure regulating zone was
about 40 °C. At this temperature, the viscosity of water decreases to 0.653 mPa·s from 1.00 mPa·s
(at 20 °C), which will make the pressure in the sterilization zone reduced from 539 kPa (calculated
at 20 °C and flow rate of 4 nl/s) to 387 kPa. The boiling point will then decrease to 142.8 °C,
which will guarantee a boiling-free sterilization. In the cases without the thermal isolation
trenches, the temperature of the pressure regulating zone reached as high as 75 °C because of the
thermal interference from the sterilization zone, as shown in Figure Figure3.3. The pressure in the sterilization zone was then reduced to 268 kPa (calculated at flow
rate of 4 nl/s) and the boiling temperature was around 130 °C, which was lower than the set
sterilization temperature. Detail calculation can be found in supplementary S2.16Open in a separate windowFIG. 3.The temperature distribution of the chips (before packaged) with and without thermal isolation
trenches (powered at 1 W). The data were extracted from the central lines of infrared images, as
shown as inserts.Bacterial sterilization performance of the present chip was tested and the experimental results
were shown in Figure Figure4.4. E. coli with initial
concentration of 106/ml was pumped into and flew through the chip with the sterilization
temperatures varied from 25 °C to 120 °C at flow rates of 2 nl/s and 4 nl/s. The outflow was
collected and inoculated onto the SS agar plate evenly with inoculation loops. The population of
bacteria in the outflow was counted based on the bacterial colonies after incubation at 37 °C for
12 h. Typical bacterial colonies were shown in Figure Figure4.4. The
low flow rate case showed a better sterilization performance because of the longer staying period in
the sterilization channel. The population of E. coli was around
1.25 × 104/ml after a 432 s-long, 70 °C sterilization (at flow rate of 2 nl/s). While at
the flow rate of 4 nl/s, the cultivation result indicated the population was around
3.8 × 104/ml because the sterilization time was shorten to 216 s. A control case, where
the solution flew through an un-heated chip at 2 nl/s, was conducted to investigate the effect of
the shear stress on the sterilization performance (see the supplementary S3 for details16). As listed in Table TableI,I, the results indicated that the shear stress did not show any noticeable effect on the
bacterial sterilization. When the chip was not heated, i.e., the case with the largest shear stress
because of the highest viscosity of fluid, the bacterial cultivation was nearly the same as the
off-chip results (no stress). The temperature has the most significant effect on the sterilization
performance. No noticeable bacteria proliferation was observed in the cases with the sterilization
temperature higher than 100 °C, as shown in Figure Figure44.
Open in a separate windowaData in the table are shear stress (Pa)/population of bacteria, where “+++” indicates a large
proliferation, “+” means small but noticeable proliferation, “−” represents no proliferation.bOff-chip control group.Open in a separate windowFIG. 4.Sterilization performance of the present chip with E. coli and S.
aureus as test bacteria. All the original population was 106/ml. Inserted images
showed the images of the culture disk after bacteria incubation.Sterilization of another commonly encountered bacterium, Staphylococcus aureus,
with initial population of 106/ml was also tested in the present chip, as shown in Figure
Figure4.4. Similarly, no noticeable S. aureus
proliferation was found when the sterilization temperature was higher than 100 °C.In short, we demonstrated a microfluidic sterilization strategy by utilizing a continuous flowing
high temperature/pressure chip. The population of E. coli or S.
aureus was reduced from 106/ml to an undetectable level when the sterilization
temperature of the chip was higher than 100 °C. The chip holds promising potential in developing
portable microsystem for biological/clinical applications. 相似文献
Table I.
The E. coli cultivation results under different flow rates and sterilization temperatures. a25 °C | 70 °C | 100 °C | 120 °C | 25 °C b | |
---|---|---|---|---|---|
2 nl/s | 1.89/+++ | 1.38/+ | 1.16/− | 1.04/− | 0/+++ |
4 nl/s | 3.78/+++ | 2.76/+ | 2.32/− | 2.08/− | 0/+++ |
6.
Muhammet Kamali 《Journal of The Franklin Institute》2007,344(6):867-872
A certain differential operator Dn+p is introduced for functions of the form
7.
8.
M.K. Aouf 《Journal of The Franklin Institute》2010,347(10):1927-1941
Let denote the class of functions analytic in U={z:|z|<1} which satisfy for fixed M, z=reiθ∈U and
9.
M.A. Bokhari 《Journal of The Franklin Institute》2007,344(5):637-645
The n-point Gauss quadrature rule states that
10.
F.B. Gao 《Journal of The Franklin Institute》2011,348(6):1020-1034
We consider an n-dimensional p-Laplacian-like neutral functional differential equation (NFDE) in the form
11.
In this paper, we consider multipoint boundary value problem for third-order differential equations with p-Laplacian at resonance
12.
Sharmila Upadhya Subramanya Upadhya D. M. Vasudevan 《Indian journal of clinical biochemistry : IJCB》2003,18(1):46-51
The study was designed to evaluate the significance of tissue polypeptide specific antigen (TPS) in patients with histologically
proven ovarian and colorectal cancer following treatment along with CA125 (in ovarian cancer) and CEA (in colorectal cancer).
Patients were grouped as follows:
In patients with ovarian and colorectal cancer, the mean TPS levels were significantly higher in patients of group II compared
to group I. The percentage of patients above cut-off levels for TPS were 17.4% in group I and 95.5% in group II. Similar results
were observed with the mean levels of CA125. In colorectal cancer patients, the percentage of patients above cut-off levels
for CEA and TPS were 70% and 30% in group I and 100% in group II for both the markers. Our observations indicate that TPS
may be used as a common marker to indicate metastases in patients with ovarian and colorectal cancer. 相似文献
Group I | : Patients with stable disease |
Group II | : Patients with metastasis and relapse |
13.
In this paper we stochastically perturb the functional Kolmogorov-type system
14.
Cemil Tunç 《Journal of The Franklin Institute》2011,348(7):1404-1415
We establish some sufficient conditions which guarantee asymptotic stability of the null solution and boundedness of all the solutions of the following nonlinear differential equation of third order with the variable delay, r(t)
15.
In this paper, the second order non-linear differential equation
16.
In this paper, we investigated the differential equation
17.
Salim A. Messaoudi 《Journal of The Franklin Institute》2007,344(5):765-776
In this paper we consider the semilinear viscoelastic equation
18.
D.N. Chalishajar 《Journal of The Franklin Institute》2007,344(1):12-21
Dhakne and Kendre [On abstract nonlinear mixed Volterra-Fredholm integro-differential equations, Presented Paper in the International Conference at IIT-Bombay, 11-13 December, 2004] has proved the existence of the abstract nonlinear mixed Volterra-Fredholm integro-differential system of the type
19.
We present a simple method for creating monodisperse emulsions with microfluidic devices. Unlike conventional approaches that require bulky pumps, control computers, and expertise with device physics to operate devices, our method requires only the microfluidic device and a hand-operated syringe. The fluids needed for the emulsion are loaded into the device inlets, while the syringe is used to create a vacuum at the device outlet; this sucks the fluids through the channels, generating the drops. By controlling the hydrodynamic resistances of the channels using hydrodynamic resistors and valves, we are able to control the properties of the drops. This provides a simple and highly portable method for creating monodisperse emulsions.Droplet-based microfluidic devices use micron-scale drops as “test tubes” for biological reactions.1, 2, 3 With the devices, the drops are loaded with cells, incubated to stimulate cell growth, picoinjected to introduce additional reagents, and sorted to extract rare specimens.4, 5, 6 This allows biological reactions to be performed with greatly enhanced speed and efficiency over conventional approaches: by reducing the drop volume, only picoliters of reagent are needed per reaction, while through the use of microfluidics, the reactions can be executed at rates exceeding hundreds of kilohertz. This combination of incredible speed and efficient reagent usage is attractive for a variety of applications in biology, particularly those that require high-throughput processing of reactions, including cell screening, directed evolution, and nucleic acid analysis.7, 8 The same advantages of speed and efficiency would also be beneficial for applications in the field, in which the amount of material available for testing is limited, and results are needed with short turnaround. However, a challenge to using these techniques in field applications is that the control systems developed to operate the devices are intended for use in the laboratory: to inject fluids, mechanical pumps are needed, while computers must adjust flow rates to maintain optimal conditions in the device.9, 10, 11, 12 In addition to significantly limiting the portability of the system, these qualities make them impractical for use outside the laboratory. For droplet-based microfluidic techniques to be useful for applications in the field, a general, robust, and portable system for operating them is needed.In this paper, we introduce a general, robust, and portable system for operating droplet-based microfluidic devices. In this system, which we call syringe-vacuum microfluidics (SVM), we load the reagents needed for the emulsion into the inlets of a microfluidic drop maker; using a standard plastic syringe, we generate a vacuum at the outlet of the drop maker,13 sucking the reagents through the channels, generating drops, and transporting them to different regions for visualization and analysis. By controlling the vacuum strength and channel resistances using hydrodynamic resistors14, 15, 16 and single-layer membrane valves,17, 18 we are able to specify the flow rates in different regions of the device and to adjust them in real time. No pumps, control computers, or electricity is needed for these operations, making the entire system portable and of potential use for field applications. To characterize the adjustability and precision of this system, we vary channel resistances and vacuum pressures while measuring the effects on drop size and production frequency. We also show how to use this to form drops of many distinct reagents simultaneously using only a single vacuum syringe.Monodisperse drop formation is the central operation in droplet-based microfluidics but can be quite challenging due to the need for precise, steady pumping of reagents; forming monodisperse drops with controlled properties is thus a stringent demonstration of the effectiveness of a control system. While there are many geometries available for microfluidic drop formation,19 in this discussion we use a simple cross-junction for its proven ability to form uniform emulsions at high rates of speed,20, 21 a schematic of which is shown in Fig. Fig.1.1. The devices are fabricated in poly(dimethylsiloxane) (PDMS) using soft lithography.22 The drop formation channels have dimensions of 25 μm in width and 25 μm in height. To enable production of aqueous drops in oil, which are the most useful for biological assays, we require hydrophobic devices, which we achieve using an Aquapel chemical treatment: we flow Aqualpel through the channels for a few seconds, flush with air, and then bake the devices for 20 min at 65 °C. After this treatment, the channels are permanently hydrophilic, as is needed for forming aqueous-in-oil emulsions. To introduce reagents into the device, we use 200 μl plastic pipette tips inserted into the channel inlets. To apply the suction, we use a 10 ml Bectin-Dickenson plastic syringe coupled to the device through a 16 G needle and PE∕5 tubing. The other end of the tubing is inserted into the outlet of the device.Open in a separate windowFigure 1Schematic of the microfluidic drop maker for use with SVM. To form water drops in oil, the device must be hydrophobic, which we achieve by treating the channels with Aquapel. The water and surfactant-containing oil are loaded into pipette tips inserted into the device inlets at the locations indicated. To pump the fluids through the drop maker, a syringe applies a vacuum to the outlet; this sucks the fluids through the drop maker, forming drops. The drops are collected into the suction syringe, where they can be stored, incubated, and reintroduced into a microfluidic device for additional processing.To begin forming drops, we fill the device with HFE-7500 fluorocarbon oil, displacing trapped air bubbles that could restrict flow and interfere with drop formation. Pipette tips containing reagents are then inserted into the device inlets, as shown in Fig. Fig.11 and pictured in Fig. Fig.2a;2a; during this step, care must be taken to not trap air bubbles under the pipette tips, as they would restrict flow. For the fluids, we use distilled water for the droplet phase and HFE-7500 with the ammonium salt of Krytox 157 FSL at 1.8 wt % for the continuous phase. The suction syringe is then connected to the device outlet; to initiate drop formation, the piston is pulled outward and locked in place with a 1 in. binder clip, as shown in Fig. Fig.2a.2a. This expands the air in the syringe, generating a vacuum that is transferred to the device through tubing. Since the inlet reagents are open to the atmosphere and thus maintained at a pressure of 1 atm, this creates a pressure differential through the device that pumps the fluids. As the fluids flow through the cross-channel, forces are generated that create drops, as shown in Fig. Fig.2b2b (enhanced online). Due to the very steady flow, the drops are highly monodisperse, as shown in Fig. Fig.2c.2c. After they are formed, the drops flow out of the device through the suction tube and are collected into the syringe. Depending on the emulsion formulation, drops may coalesce on the metal needle of the syringe; if so, an Upchurch fitting should be used to couple the tubing instead. The collected drops can be stored in the syringe, incubated, and reintroduced into additional microfluidic devices, as needed for the assay.Open in a separate windowFigure 2Photograph of the microfluidic drop formation device with pipette tips containing emulsion reagents and vacuum syringe for pumping (a). Distilled water is used for the droplet phase and HFE-7500 fluorocarbon oil with fluorinated surfactant for the continuous phase. The vacuum applies a pressure differential through the device that pumps the fluids through the drop maker (b) forming drops. The drops are monodisperse, due to the controlled properties of drop formation in microfluidics (c). The scale bars denote 50 μm (enhanced online).In many biological applications, drop size must be precisely controlled. This is essential, for example, when encapsulating molecules or cells in the drops, in which the number encapsulated depends on the drop size.3, 23, 24 With SVM, the drop size can be precisely controlled. Our strategy to accomplish this is motivated by the physics of microfluidic drop formation. In microfluidic devices, the capillary number of the flow is normally small, Ca<0.1; as a consequence, the drop formation physics follows a plugging∕squeezing mechanism, in which the drop size depends on the flow rate ratio of the dispersed-to-continuous phase.20, 25 By adjusting this ratio, we can thus control the drop size. To adjust this ratio, we use hydrodynamic resistor channels.14, 15, 16 These channels are analogous to electronic resistors in that for a fixed pressure drop (voltage) the flow rate through them (current) is inversely proportional to their resistance. By making the resistors longer or shorter, we adjust their resistance, thereby controlling the flow rate.To use resistors to control the drop size, we place three on the inlets of the cross-junction, at the locations indicated in Fig. Fig.3a.3a. In this configuration, the flow rate ratio depends on the resistances of the central and side resistors: shortening the side resistors increases the continuous phase flow rate with respect to the dispersed phase, thereby reducing the ratio and, consequently, the drop size, whereas lengthening it increases the drop size. By varying the ratio, we produce drops over a range of sizes, as shown in Fig. Fig.3b3b (enhanced online). The drop size is linear in the resistance ratio, indicating that it is linear in the flow rate ratio, as is expected for plugging∕squeezing drop formation [Fig. [Fig.3b3b].20, 25 This behavior is identical to that of pump-driven fluidics, demonstrating that SVM affords similar control.Open in a separate windowFigure 3Drop properties can be controlled using resistor channels. The resistors are placed on the inlets of the drop maker at the locations indicated in (a). The resistors enable the flow rates of the inner and continuous phases to be controlled. By varying the length ratio of the inlet resistors, we control the flow rate ratio in the drop maker. This allows the drop volume to be controlled, as shown by drop volume plotted as a function of inlet resistor length ratio in (b); varying this ratio does not significantly affect the drop formation frequency, as shown in (c). By varying the length of the outlet resistor, we control the total flow rate through the device; this allows us to form drops of constant volume, but at a different formation frequency, as shown by the plots of volume and frequency as a function of the inverse of the outlet resistor length in (d) and (e), respectively. The measured hydrodynamic resistance of a resistor channel with water as a function of length is shown as inset into (d) (enhanced online).We can also control the frequency of the drop formation using resistor channels. We place a resistor on the outlet of the device; this sets the total flow rate through the device, thereby adjusting drop frequency, as shown in Fig. Fig.3e3e (enhanced online). To confirm that the size and frequency control are independent, we plot size as a function of the outlet resistance and frequency as a function of the resistance ratio [Figs. [Figs.3c,3c, ,3d];3d]; both are constant as a function of these parameters, again demonstrating independent control. Frequency can also be adjusted by changing the strength of the vacuum, which can be accomplished by loading a prescribed volume of air into the syringe before expansion. In this case, the vacuum pressure applied is Pfin=Vin∕Vfin×Pin, where Vin is the initial volume of air in the syringe, Vfin is the volume after expansion, and Pin is the initial pressure, which is 1 atm. By loading a prescribed volume of air into the syringe before connecting it to the device and pulling the piston, the expansion factor can be reduced, thereby lowering the vacuum strength.The flow rates through the microfluidic device depend on the applied pressure differential, which, in turn, depends on the value of the ambient pressure. Since ambient pressure may vary due to differences in altitude, the drop formation may also vary. However, since ambient pressure variations affect the inner and outer phase flows equally, this should alter the total flow rate but not the flow rate ratio. Consequently, we expect it to alter drop formation frequency but not drop size because while the frequency depends on absolute flow rate [as illustrated by Fig. Fig.3e],3e], drop size depends on the flow rate ratio [as illustrated in Fig. Fig.3b].3b]. Based on normal variations in atmospheric pressure on the surface of the Earth, we expect this to produce differences in the drop formation frequency of ∼25%, for example, when operating a device at sea level compared to at the top of a moderately sized mountain.Resistor channels allow drop properties to be controlled, equivalent to what is possible with pump-driven flow; however, they do not allow real-time control because their dimensions are fixed during the fabrication. Real-time control is often needed, for example, as it is when performing reactions in drops for the first time, in which the optimal drop size is not known. To enable real-time control, we must adjust flow rates, which can be achieved using the fluidic analog of electronic potentiometers. Single-layer membrane valves are analogous fluidic components, consisting of a control channel that abuts a flow channel.17, 18 By pressurizing the control channel, the thin PDMS membrane between these channels is deflected laterally, constricting the flow channel, thereby increasing its hydrodynamic resistance and reducing its flow rate.18 To use these membrane valves to vary drop size, we replace the inlet resistors with inlet valves, as shown in Fig. Fig.4a.4a. To set the flow rate through a path, we actuate the valve with a defined pressure. To actuate the valves, we use air-filled syringes: a 1 ml syringe is filled with air and connected to the valve control channel through tubing; an additional component, a three-way stopcock is inserted between the syringe and needle, allowing the pressure to be locked in after optimal actuation conditions are obtained. We use one syringe to control the dispersed phase valves and another to control the continuous phase valves. The valves are pressurized by compressing the air in the syringes to a defined degree using the marked graduations; this is achieved by pressing the piston to a defined graduation mark, compressing the air contained within it, thus increasing pressure. The stopcock is then switched to the off position, locking in the actuation. This simple scheme allows precise actuation of the valves, for accurate, defined flow rates in the drop maker, and controlled drop size, as shown in Figs. Figs.4b,4b, ,4c4c (enhanced online). The drop size can be varied at a rate of several hertz without noticeable loss of control; moreover, changing the drop size does not affect the frequency, indicating that, again, these properties are independent, as shown by the constant drop frequency with varying pressure ratio in Fig. Fig.4d4d.Open in a separate windowFigure 4Single-layer membrane valves allow the drop size to be varied in real time to screen for optimal reaction conditions. The valves are positioned on the inner and side inlets, as indicated in (a). By adjusting the actuation pressures of the valves, we vary the flow rates in the drop maker, thereby changing the drop size (b), as shown by the plot of drop volume as a function of the actuation pressure ratio in (c). Varying the inlet resistance ratio does not significantly alter drop formation frequency, as shown by frequency as a function of the pressure ratio in (d). A movie of drop formation during actuation of the valves are available in the supplemental material (Ref. 29). The scale bars denote 100 μm (enhanced online).Another useful attribute of SVM is that it readily lends itself to parallel drop formation26 because the pressure that pumps the fluids through the channels is supplied by the atmosphere and is applied evenly over the whole outer surface of the device. This allows fluids to be introduced at equal pressures from different inlets, for forming drops with identical properties in different drop makers. To illustrate this, we use a parallel drop formation device to emulsify eight distinct reagents simultaneously; the product of this is an emulsion library, consisting of drops of identical size in which different drops encapsulate distinct reagents, useful for certain biological applications of droplet-based microfluidics.7 The microfluidic device consists of eight T-junction drop makers.25 The drop makers share one oil inlet and outlet but each has its own inner-phase inlet, as shown in Fig. Fig.5.5. The oil and outlet channels are wide, ensuring negligible pressure drop through them, so that all T-junctions are operated under the same flow conditions. A distinct reagent fluid is introduced into the inner phase of each T-junction, for which we use eight concentrations of the dye Alexa Fluor 680 in water. After loading these solutions into the device through pipette tips, a syringe applies the vacuum to the outlet, sucking the reagents through the T-junctions, forming drops, as shown by the magnified images of the T-junctions during drop formation in Fig. Fig.5.5. Since the drop makers are identical and operated under the same flow conditions, the drops formed are of the same size, as shown in the magnified images in Fig. Fig.55 and in a movie available in the supplemental material.29Open in a separate windowFigure 5Parallel drop formation device consisting of eight T-junction drop makers. The drop makers share a common oil inlet and outlet, both of which are wide to ensure even pressure distribution to all drop makers; support posts prevent these channels from collapsing under the suction. Each drop maker has its own inner-phase inlet, allowing emulsification of a distinct reagent. Since the drop maker dimensions and pressure differentials are constant through all drop makers, the drops formed are of the same size, as shown in the magnified images. The drops are ∼35 μm in diameter.To verify that the dye solutions are successfully encapsulated, we image a sample of the collected drops with a fluorescent microscope. The drops are confined in a monolayer between two glass plates so they can be individually imaged. They are of the same size but have distinct fluorescence intensities, as shown in Fig. Fig.6a.6a. To quantify these differences, we measure the intensity of each drop and plot the results as a histogram [see Fig. Fig.6b].6b]. There are eight peaks in the histogram, corresponding to the eight dye concentrations, demonstrating that all dyes are encapsulated successfully. The peak areas are also similar, demonstrating that drops of different types are formed in equal amounts due to the uniformity of the parallel drop formation.Open in a separate windowFigure 6Fluorescent microscope image of emulsion library created with parallel T-junction device (a). In this demonstration, eight concentrations of Alexa Fluor 680 dye are emulsified simultaneously, producing an emulsion library of eight elements. The drops are of the same size but encapsulate distinct concentrations of the dye solution, as demonstrated by the eight peaks in the intensity histograms in (b). The scale bar denotes 100 μm.SVM is a simple, accessible, and highly controlled way to form monodisperse emulsions for biological assays. It allows controlled amounts of different reagents to be encapsulated in individual drops, drop size to be precisely controlled, and the ability to form drops of different reagents at the same time, in a parallel drop formation device. These properties should make SVM useful for biological applications of monodisperse emulsions;1, 2, 3 the portability of SVM should also make it useful for applications in the field, particularly when no electrical power source is available. The parallel emulsification technique should also be useful for particle templating from drops, in which the particles must be of the same size but composed of distinct materials.26, 27, 28, 29 相似文献
20.
Plasmonic hot spots, generated by controlled 20-nm Au nanoparticle (NP) assembly, are shown to suppress fluorescent quenching effects of metal NPs, such that hair-pin FRET (Fluorescence resonance energy transfer) probes can achieve label-free ultra-sensitive quantification. The micron-sized assembly is a result of intense induced NP dipoles by focused electric fields through conic nanocapillaries. The efficient NP aggregate antenna and the voltage-tunable NP spacing for optimizing hot spot intensity endow ultra-sensitivity and large dynamic range (fM to pM). The large shear forces during assembly allow high selectivity (2-mismatch discrimination) and rapid detection (15 min) for a DNA mimic of microRNA.Irregular expressions of a panel of microRNAs (miRNA) in blood and other physiological fluids may allow early diagnosis of many diseases, including cancer and cardiovascular diseases.1 However, quantifying all relevant miRNAs (out of 1000), with similar sequences over 22 bases2 and large variations in expression level (as much as 100 fold) at small copy numbers, requires a new molecular diagnostic platform with high-sensitivity, high-selectivity, and large dynamic range. Current techniques for miRNA profiling, such as Northern blotting,3 microarray-based hybridization,4 and real-time quantitative polymerase chain reaction5 are expensive and complex. A simple and rapid miRNA array would allow broad distribution of molecular diagnostic devices for cancer and chronic diseases, eventually into homes for frequent prescreening of many diseases.At their low concentrations in untreated samples, optical sensing of miRNA is most promising. Plasmonically excited Raman scattering (SERS) and fluorescence sensors from metallic nanoparticles (NPs) or surfaces have enhanced the sensitivity of optical molecular sensors by orders of magnitude.6, 7, 8, 9 However, probe-less SERS sensing or fluorescent sensing of unlabeled targets are insufficiently specific for miRNA targets in heterogeneous samples. Plasmonic detection is also very compatible with FRET probes whose donor dye offers small light sources to excite fluorescently labelled targets upon hybridization.7, 10A particular family of FRET reporters does offer label-free sensing: hairpin oligo probes whose end-tagged fluorophores are quenched by the Au NP to which they are functionalized.11 The fluorescent signal is only detected when the hairpin is broken by the hybridizing target nucleic acid or protein (for an aptamer probe), and the more rigid paired segment separates the end fluorophore from the quenching surface to produce a fluorescent signal. It is often hoped that plasmonics on the metal surface will enhance the intensity to overcome the quenching effect, if the linearized hairpin is within the NP plasmonic penetration length. However, since fluorescent quenching decays slowly (linearly) with fluorophore-metal spacing10 whereas the plasmonic intensity decays exponentially from a flat surface, careful experimentation shows that quenching dominates and the hairpin probe actually produces a larger intensity on non-metallic surfaces,10 on which it can not function as a label-free probe. Hence, only μM limit-of-detection (LOD) has been achieved with this technique on single NPs or on flat metal surfaces,12 with expensive laser excitation and confocal detection.Plamonic hot spots formed between metal nanostructures and sharp nanocones can further amplify the plasmonic field.13, 14 The hot spot intensity decays algebraically with respect to the separation or cone tip distance and hence should dominate the linear decay of the metal quenching effect at some optimum separation.15 It is hence possible that plasmonic hot spots may allow much lower LOD with inexpensive optical instruments—ideally light-emitting diode light source and miniature camera. However, the dimension of the gaps, cones, and wedges needs to be at nanoscale, and the cost is now transferred to fabrication of such hot-spot substrates like bow-ties, double crescents, bull-eyes, etc.16 Low-cost wet-etching techniques for addressable nanocones that sustain converging plasmonic hot spots17 have been reported but the fabricated nanocones are often too non-uniform to allow precise quantification. NP monolayers have been shown to exhibit plasmonic hot spots and fluorescence enhancement.18, 19 However, the enhancement only occurs within a range of spacing between aggregated NPs, which is difficult to control and the location or even the existence of the hotspots are not known a priori.Higher sensitivity is expected if a minimum number of NPs are used in an assembly at a known location and if the NP assembly can produce crystal-like aggregates with controllable NP spacing. Induced DC and AC NP dipoles (related to dielectrophoresis) have been used to assemble NP crystals by embedded micro-electrodes to provide the requisite high field.20, 21 The resulting NP crystals are ideal for plasmonic hot spots, since the spacing of the regimented NP crystal can be controlled by the applied voltage. Conic nanocapillaries22, 23 will be used here for such field-induced NP assembly because the submicron-tip can focus the electric field into sufficient high intensity for NP assembly without embedded-electrodes. Because the field is highest at the tip due to field focusing, the micron-sized crystal would be confined to a small volume, which will be shown to be less than typical confocal volumes, at a known location. So long as the hotspots are regimented, the quantification of target molecules is determined by the total fluorescent intensity and is hence insensitive to the exact geometry of the nanocapillary.Fluorescent microscope equipped with tungsten lamp light source and normal CCD camera from Q Imaging were used for simultaneous optical and ion current measurements, as shown in Fig. Fig.1a.1a. The nanocapillaries were pulled from commercial glass capillaries using laser-assisted capillary puller. SEM image of a typical pulled glass nanocapillary in Fig. Fig.1b1b shows an inner diameter of 111 nm and cone angle of 7.3°. The capillary was inserted into a Polydimethylsiloxane chip with two reservoirs. The 20 nm Au NPs, functionalized with fluorescently labelled dsDNA, were injected into the base reservoir. With SEM imaging (Fig. S3 in the supplementary material24), the functionalized DNA is found to prevent NP aggregation even in high ionic-strength Phosphate buffered saline buffer. The NP solution is then driven into the capillary through the tip by applying a positive voltage. Fig. Fig.1c1c shows the ion current evolution over 2 h at +1 V packing voltage. The ion current increases rapidly in the first 10 min, then at a much slower rate. The rise of current indicates assembly of conductive Au NP assembly at the tip. This was confirmed by the strong fluorescence signal at the tip region during the packing process (inset of Fig. Fig.1c).1c). The one-micron region (corresponding to roughly an aggregate volume of one attoliter) near the capillary tip shows a fluorescence signal after 1 min and also appeared as a dark spot in the transmission image (supplementary material, Fig. S124). This spot darkens with longer packing time but does not grow in size, consistent with the monotonically increasing ion current with increased packing density of the NP assembly. As contrast, a strong fluorescence appeared after only 1 min of packing, but the signal became weaker after 15 min (supplementary material, Fig. S124). This reduction in fluorescence is not due to bleaching of fluorophores because we took 2 images in 15 min at 5 s exposure each and control experiments show significant bleaching only beyond an exposure time of 100 s (see supplementary material).24 Instead, the non-monotonic dependence of the fluorescence intensity with respect to time is because of the optimal hotspot spacing for highest plasmonic intensity at about 5–20 nm,25, 26, 27 which is reached at about 10 min.Open in a separate windowFigure 1Plasmonic hotspots generated at the tip of a nano-capillary. (a) Schematic of the experimental set up. (b) SEM image of glass nanocapillary shows opening at the tip with a diameter of 111 nm. (c) Current evolution during packing of fluorescently labeled gold particles at +1 V. Inset shows strong fluorescence only after 1 min of packing.The FRET probe is designed to exploit the plasmonic hotspot.24 We first electrophoretically drove the target molecules in the tip side reservoir into the nano-capillary by applying a negative voltage of −1 V. During this process, the targets are trapped within the capillary and hybridize with the hairpin probes on the Au NP in the nanocapillary. Fluorescence of the unquenched hybridized probes is too weak to be detected by our detector as shown in Fig. Fig.2b.2b. A reverse positive voltage of +1 V was then applied to the capillary to pack the Au NPs to the tip. Due to plasmonic hot spots of aggregated gold nanoparticles, the fluorescence signal is significantly enhanced at the tip and can be detected by our CCD camera, as shown in Fig. Fig.2c2c.Open in a separate windowFigure 2(a) Schematics of designed hairpin probe on gold particle. (b) Before packing gold particles, probe fluorescence signal was too weak to be detect. (c) After packing for 3 minutes, a strong fluorescence signal appears at the NP aggregate. (d) Normalized intensity (average of all pixels above a threshold (15 au) normalized with respect to the average over all pixels (with 0-250 au)) as a function of packing voltage for different samples. Black, 1 nM target ; blue, 10 pM target; purple, 10 nM 2-mismatch non-target. (e) Intensity dependence on target concentration. Measured normalized intensity before packing (black) and after packing (red), for three independent experiments with different nano-capillaries at each concentration. NT stands for non-target at 10 nM as a reference.For the same packing time, the fluorescence intensity increases initially but saturates after 10 min time of trapping (supplementary material, Fig. S2(a)24). Over 10 min of trapping with a negative voltage, we found the fluorescence intensity exhibits a maximum at a packing time of 3 min (supplementary material, Fig. S2(b)24). In later experiments, we used 10 min trapping time and 3 min packing time as standards.Fig. Fig.2d2d shows the fluorescence intensity is sensitive to the positive packing voltage at different concentration of target and non-target molecules. For target samples (1 nM and 10pM), the optimal voltage is about 1 V. We suspect that with larger voltage, the NPs are packed too tightly such that the NP spacing is smaller than the optimal distance for plasmonic hotspots. The fluorescence intensity for a nontarget with two mismatches is 7 times lower than the target even with a 10 times higher concentration (10 nM). Moreover, the optimal voltage for the non-target miRNA is reduced to 0.5 V instead 1 V for the target miRNA. Strong shear during electrophoretic packing has probably endowed this high selectivity.20Using the protocol above, the LOD and dynamic range of the target was determined (Fig. (Fig.2e).2e). The intensity at each concentration is measured with three independent experiments with different nanocapillaries to verify insensitivity with respect to the nanocapillary. The intensity increases monotonically with respect to the concentration from 1fM to 1pM. Beyond 1pM, the fluorescence signal saturates, presumably because all hairpin probes at the tip have been hybridized. At 1 fM, the fluorescent intensity is still well above the background measured from the non-target sample. Note both auto-fluorescence of gold nanoparticles and free diffusing non-target DNA molecules contribute to the background. Given the volume of tip side reservoir (∼50 μl), there are about 30 000 target molecules in the reservoir at 1 fM. However, with a short 10 min trapping time, we estimate only a small fraction of these molecules, less than 100, have been transferred from the tip reservoir into the nanocapillary. 相似文献