TY - JOUR
AU - Pelssers,, Eduard
AB - Abstract Constructs consisting of a channel, a membrane, and an absorber are designed for autonomously carrying out various liquid-handling functions of analytical tests. These so-called fluid elements can be used to set up various circuits for conducting several kinds of analytical tests. To demonstrate the feasibility of this concept, we constructed such a circuit and used it to perform, with two handling steps, an ELISA of hepatitis B surface antigen. The detection limit of the assay was comparable with those of state-of-the-art ELISAs for screening blood, and results could be obtained within a total test time of 20 min. We anticipate that this concept of automation may also serve as a basis for new, highly simplified immunoanalyzers. indexing terms: ELISA, hepatitis B surface antigen, immunoanalyzers Numerous assays have been developed for qualitative and quantitative detection of various analytes used in the biochemical diagnosis of human and animal disorders. Assay developers are continually trying to make these assays easier to perform and thus more available for use by nontechnical personnel in a wide variety of environments, e.g., doctors’ offices, clinics, patients’ homes, crisis centers, emergency rooms, ambulances, blood banks, and hospital laboratories. Toward achieving these improvements, various different concepts of automation have been applied to diagnostic tests. For example, continuous-flow analyzers (1)(2) have been used as clinical chemistry analyzers but are not suitable for immunological tests because of those tests’ stringent requirements with respect to carryover. Especially for immunologically based tests, robotics is used in instrumental analyzers that process separate cuvettes. Often, the physical procedural steps previously carried out by technicians are copied by robotics; for single tests, however, robotic automation is obviously unsuitable. Consequently, different concepts of automation have been developed. For example, in a dipstick pregnancy test (Predictor; Chefaro International, Rotterdam, The Netherlands), a porous carrier containing all reagents is wetted by urine, and the analytical reactions are conducted during the subsequent transport of the urine by the capillary action of the porous carrier; particles incorporated in the carrier are used to create a physically detectable signal. Performance of these tests is analogous to the action of a continuous-flow analyzer: movement of liquid through a defined track progressing in one direction. Here we introduce a new concept for transporting liquid in a timely manner in various directions; the resulting liquid flow is more complicated than that achieved in a dipstick test. The concept is based on so-called fluid elements, constructs that can be used to conduct various well-defined liquid-handling functions (i.e., processes typically taking place during execution of an analytical test). Connecting these elements in various ways yields different circuits, each capable of carrying out different types of analytical tests. To demonstrate the feasibility of the concept, we constructed a circuit of fluid elements to conduct an ELISA that has a relatively complicated test protocol. In the circuit of elements presented here, the main parts of the transport functions of the assay are carried out autonomously by the fluid elements. In contrast, ELISAs in microtiter plate format require the various handling steps to be carried out manually (e.g., pipetting liquids and moving the plates to the incubator, the washer, and the reader). An important advantage of the manual test format is its design to have no substantial increase in the number of handling steps with respect to a dipstick test but to be more sensitive through the use of an enzyme label. The further development of fluid elements may allow a circuit of elements to be used as a disposable “cuvette” in an immunoanalyzer and thereby largely obviate the need for robotics. Theory liquid transport in the fluid element In principle, a fluid element consists of a channel bordered partially with a semipermeable membrane, with an absorbent material (absorber) placed on the other side, as schematically depicted in Fig. 1 . The element has three gates, each of which can be used to connect it to a circuit of elements. Beside the entrance and the exit of the channel, a third gate is formed by an air vent coupled directly to the absorber area. The liquid flux of the liquid entering the channel (Q1) is governed by the (applied) hydrostatic pressure (ρgz, where ρ is the liquid density, g the gravitational acceleration, and z the height of the liquid column) and the capillary pressure of the channel such that: \[\mathrm{Q}_{\mathrm{1}}\mathrm{\ {=}\ ({\rho}}gz{+}2{\gamma}\mathrm{cos{\theta}}_{\mathrm{adv}}\mathrm{/}D)/(12{\mu}h(t)/D^{3}W{+}\mathrm{R}_{\mathrm{cir}}\mathrm{)}\] The term 2γcosθadv/D is the capillary pressure difference at the liquid front according to the Laplace equation for a slit geometry (where the depth D is much smaller than the width W). The surface tension of the liquid is represented by γ, and θadv is the advancing contact angle at the liquid front. The term (12 μh(t)/D3W) is the hydrodynamic resistance of a slit according to the Poiseuille equation, with h(t) the distance between the entrance and the position of the liquid front at time t; μ is the viscosity of the liquid; and Rcir is the hydrodynamic resistance of the fluid circuit in front of the element. In the first stage, when the liquid has not yet reached the membrane, the contact angle θ will adapt to the value of the advancing contact angle θadv. In the second stage, the membrane is wetted and subsequently so is the absorber. During this transitional process, no substantial liquid flow into the absorber is developed and the liquid front continues to progress into the channel. Only thereafter is the third stage reached, in which a liquid flux Q2 through the membrane into the absorber is established by the capillary action of the absorber. The aspiration power of the absorber can be characterized by the initial value of Q2 (see Fig. 1 caption). If the absorber has a low aspiration power, the liquid front will continue to progress through the channel and, consequently, more membrane surface will be wetted. Therefore, the hydrodynamic resistance to flow into the absorber will decrease and Q2 will increase. If in this case Q2 matches Q1, the liquid front will come to a halt, with θ ≈ θadv. For an absorber with medium aspiration power, the initial flux Q2 will decrease the pressure at the meniscus of the liquid front in the channel; consequently, Q2 will decrease and Q1 will increase until both are matched at the same value. An absorber with a large aspiration power will decrease the pressure at the liquid site of the meniscus to even less than the capillary receding pressure, and the liquid front in the channel will start to retreat; consequently, the membrane surface available for liquid transport into the absorber will decrease, and the hydrodynamic resistance for liquid flow into the absorber will therefore increase. As a result, the liquid front in the channel again will halt, and Q1 and Q2 will be regulated to exactly the same value, with θ ≈ θrec. In the cases where Q1 = Q2, the value of the fluxes during the filling of the absorber can be calculated as: \[\mathrm{Q}_{\mathrm{1}}\mathrm{\ {=}\ Q}_{\mathrm{2}}{=}({\rho}gz{+}2{\gamma}\mathrm{cos}{\theta}/D)/(12{\mu}h_{\mathrm{eq}}/D^{3}W{+}\mathrm{R}_{\mathrm{cir}}\mathrm{)}\] where heq is the distance between the entrance and the position of the stationary liquid front. The actual aspiration power of an absorber is determined by several factors: the capillary pressures in the absorber, the flow resistance of the absorber (depending on porosity and shape), and the flow resistance of the membrane (depending on the pore size, pore density, and surface area used for transmembrane flow). After the absorber is saturated with liquid, the fourth and last stage is reached and the liquid front in the channel starts to move again toward the exit of the element with a flux Q3 = Q1, according to Eq. 1. The time to fill the absorber, δTabs, can be calculated by: \[{\delta}T_{\mathrm{abs}}{=}V_{\mathrm{abs}}\mathrm{Por/Q}_{\mathrm{2}}\] where Vabs is the volume of the absorber and Por is the porosity factor of the absorber (fraction of volume available for liquid). The time to fill the channel, δTch, can be derived from the Washburn equation (3), as adapted for a slit geometry: \[{\delta}T_{\mathrm{ch}}{=}DW{[}(12{\mu}/D^{3}W)\ 1/2h_{\mathrm{ele}}^{\mathrm{2}}\mathrm{{]}}\] \[{+}\ R_{cir}h_{\mathrm{ele}}\mathrm{\}/(2{\gamma}cos{\theta}}_{\mathrm{adv}}\mathrm{/}D{+}{\rho}gz)\] where hele is the length of the channel in the element. The time delay, δT, of liquid entering the element and exiting the element is a summation of δTabs and δTch. The time taken by the wetting process in the second stage is neglected because, in the experimental setups, this time is short with respect to the time needed to fill the absorber. separation process in the element One of the applications of the element is to separate species of different sizes. The membrane separates species contained in the liquid by size exclusion. This process is conducted by leading a liquid sample, containing the species to be separated, into the element and subsequently adding a wash liquid to the element. After the sample is absorbed, residual layers of the liquid sample will still remain in the channel. These nonremoved residual layers contain all components in the concentrations originally present in the liquid sample, and these components will migrate into the wash liquid only by passive diffusion. The fraction of the components α left in these residual layers after a wash time of t can be correlated to the thickness of the layer δ and the diffusion coefficient (Dc) of the component under consideration (4): \[{\alpha}{=}\mathrm{exp({-}D}_{\mathrm{c}}t/{\delta}^{2})\] After the absorber is saturated, the wash liquid will change direction from migrating into the absorber toward migrating in the direction of the exit. From that moment on, the components diffusing into the wash liquid will exit the element, and the concentration in the liquid, being proportional to α, will therefore decrease exponentially with time according to Eq. 5. Both Dc and δ are intrinsic parameters of the assay and can be improved only by changing the assay conditions. In the design of the element, the surfaces used mutually by assay mix and wash liquid should be minimized and sufficient time should be allowed for diffusion to occur (this time is proportional to the size of the absorber). liquid transport by the element Another application of the element is to transport liquid samples. During liquid uptake by the absorber, the air originally present in the absorber is displaced. When the air vent is connected to a chamber where a liquid sample is positioned, this liquid sample can be transported to another location by the air pressure building up between the element and the chamber. The minimum pressure (Pbar) to start displacing a liquid sample depends on the pressure barrier caused by the hysteresis effect of the capillary pressure at the advancing and receding meniscus of the liquid sample. For a chamber with circular cross-section, this pressure barrier can be calculated as follows: \[P_{\mathrm{bar}}{=}\mathrm{4{\gamma}cos({\theta}}_{\mathrm{rec}}\mathrm{{-}{\beta})/}D_{\mathrm{rec}}\mathrm{\ {-}\ 4{\gamma}cos({\theta}}_{\mathrm{adv}}{+}{\beta})/D_{\mathrm{adv}}\] where θrec is the receding contact angle and Drec is the diameter of the chamber at the position of the receding meniscus; θadv represents the advancing contact angle and Dadv is the diameter at the position of the advancing meniscus; and β represents the inclination angle of the surface of the chamber. In many cases, a chamber with equal cross-sectional diameters will be used, such that Dadv = Drec and β = 0. However, in the case Dadv ≪Drec, the pressure barrier changes into a pressure that forces the liquid sample to migrate spontaneously. The maximum pressure (Pmax) that can be built up by the absorber equals the pressure at which the liquid is pressed out of the largest pores of the absorber: \[P_{\mathrm{max}}{=}4{\gamma}\mathrm{cos{\theta}}^{\mathrm{abs}}_{\mathrm{rec}}\mathrm{/}D_{\mathrm{max}}\] where θabsrec is the receding contact angle of the absorber, and Dmax is the diameter of the largest pores of the absorber. In the case where Pmax ≫ Pbar, then Q4, the flux of the liquid sample, equals Q2. supporting elements Beside the fluid elements, three supporting elements are used. These elements perform the functions of interfacing the circuit of elements to the outside world: providing liquid sample, providing wash liquid, providing a hydrostatic pressure, and rendering a physically detectable signal. Materials and Methods materials The membrane is a track-etched membrane of poly(ethylene terephthalate) (Cyclopore; Whatman SA, Louvain La Neuve, Belgium), which has well-defined orthogonal transmembrane pores; its hydrophilicity is regulated by a proprietary method. Descriptive data provided by the manufacturer include: average pore density, 3.6 × 107 pores/cm2; thickness, 11 μm; transmembrane flux, 103 mL • min−1 • cm−2 at 10 psi (∼69 kPa); and average pore diameter, 0.68 μm. The glass-fiber absorber was purchased from Whatman, Maidstone, UK. Using a porometer (Coulter, Mijdrecht, The Netherlands) and Coulter Porofill as test liquid, we determined the pore distribution. Measured characteristics are: average pore size, 3.8 μm; pore size range, 2.3–10.9 μm; porosity, 0.95; and thickness, 0.8 mm. The channel is positioned in an injection-molded polycarbonate material (Lexan R144; General Electric, Bergen op Zoom, The Netherlands). No grease or other lubricants were used during injection-molding, and the parts were cleaned with absolute ethanol (Merck, Darmstadt, Germany) and dried at ambient temperature before use. Dimensions of the channel are 2 ± 0.02 mm wide and 100 ± 5 μm deep; the channel length is a few centimeters, depending on the specific element. The cover part, in which the absorber-cavity is placed, is made and treated in the same manner. elisa reagents The solid phase consists of 811-nm-diameter monodisperse red polystyrene latex particles (laboratory sample AEX7 red, as prepared by corporate research of AKZO Nobel, Arnhem, The Netherlands) to which 2.5 mg/m2 F(ab′)2 fragments of a monoclonal antibody (laboratory sample OT IgG 2D; Organon Teknika, Boxtel, The Netherlands) against hepatitis B surface antigen (HBsAg) have been adsorbed. The final concentration of latex in the assay is 0.2 g/L. The conjugate consists of a sheep-anti-HBsAg polyclonal antibody (Organon Teknika) conjugated to horseradish peroxidase (HRP) from Boehringer Mannheim (Mannheim, Germany). The final conjugate concentration in the test is 12.5 μg/mL. On average, 4.4 HRP molecules are bound to one antibody molecule. Test samples were made by adding to pooled normal human serum (a mixture of at least 10 individual sera) HBsAg secreted from the primary liver carcinoma cell line PLC/PRF/5 produced in protein-free hollow fiber culture (PLC-HBsAg; Organon Teknika). The substrate compounds are stored in a matrix of nylon tortuous membranes, pore size 0.2 μm (MSI Europe, Bergen op Zoom, The Netherlands). This matrix is used in a supporting element, in which the substrate reaction is carried out. The tortuous membranes are wetted by dipping into a substrate solution, after which excess liquid is removed by pressing the membranes between two rollers. This matrix is further dried under preheated nitrogen (30 °C) for 20 min. Membranes are stored under nitrogen at 4 °C in sealed aluminum sachets containing dry silica gel. Two membranes are used per element—one impregnated with a substrate solution containing 3,3′,5,5′-tetramethylbenzidine dihydrochloride (pro analyze; Fluka, Neu-Ulm, Germany), the second impregnated with a substrate solution containing hydrogen peroxide. methods Assembly of elements (and circuits of elements). Membranes are sealed on the channel by a stamp that is heated to 238 ± 1 °C and exerts a pressure of 200 ± 10 kPa for 3 ± 0.1 s. Contact between absorber and membrane is maintained reproducibly by placing the absorber against the membrane with a slight pressure. This pressure is provided by placing the absorber in a cavity 0.7 ± 0.02 mm deep (the noncompressed absorber is 0.8 ± 0.02 mm thick). The injection-molded parts, one containing the cavity and the other containing the channel, are connected by heat-sealing at certain positions or by using nuts and bolts (Fig. 2 ). The dimensions of the experimental elements are optimized for performing the functions to be carried out (see Figs. 3 and 4). The fluid element that provides for the separation of the bound and unbound conjugate molecules is called the bound/free element. The fluid element transporting the assay mix by air pressure is called the pressure element, and the fluid element that delays the liquid transport and thereby controls the incubation time of the assay is called the timing element. The supporting element that accepts the sample or assay mix is called the input element. The supporting element that provides the substrate reaction is called the output element, and the supporting element providing hydrostatic pressure is called the hydrodynamic power source. Measurement of physical parameters. The latex recovered from a bound/free element is defined as the percentage of solid phase recovered in the first 40 μL of liquid exiting the element with respect to the amount initially added. The efficiency of the separation of bound and unbound conjugate molecules in a bound/free element is defined as the percentage of unbound conjugate molecules that is not recovered in the first 40 μL of the liquid exiting the element. The values for the physical parameters were experimentally determined; details are available upon request. Test of assay reagents and procedure. We tested the reagents independently of the fluid elements as follows. We mixed 10 μL of latex solid phase (8.5 g/L) with 90 μL of normal human serum containing 10 mL/L normal sheep serum in a 1.5-mL Eppendorf micro test tube (Eppendorf, Hamburg, Germany). After adding 100 μL of conjugate solution and mixing, we added 200 μL of test sample, mixed, and incubated the assay mix solution for 5 min at ambient temperature. The reaction was stopped by adding 4 μL of sheep-anti-HBsAg polyclonal antibody (Organon Teknika), 30 g/L. Using this much excess antibody ensures that all free binding sites on the HBsAg will be occupied by IgGs and that no more conjugate molecules can bind to the antigen during the rest of the procedure. Subsequently, we washed the latex solid phase by mixing it with 1 mL of wash buffer [50 mmol/L glycine, pH 9 (Janssen Chimica, Geel, Belgium), and 1 g/L bovine serum albumin HR (Organon Teknika)]. We then separated the particles by centrifugation (15 000g, 5 min) and redispersed the pellet in 1 mL of wash buffer by vortex-mixing. After repeating this wash procedure for a total of four times, we resuspended the solid phase in 80 μL of water, pipetted 7.5 μL of this dispersion into a microtiter well, and added 100 μL of substrate solution from the Hepanostika Uni-form II 1.0 kit for HBsAg (Organon Teknika) to the well. After incubation for 5 min, the substrate reaction was stopped by the addition of 100 μL 1 mol/L H2SO4 (Baker, Deventer, The Netherlands). To measure the absorption of the product at 450 nm, we used a Model 510 microtiter plate reader from Organon Teknika, Turnhout, Belgium. Assay performed with circuits of fluid elements. The integrated circuit performs an assay as follows (Fig. 5 ): The assay mix, prepared as described above, is incubated for 3 min, after which 20 μL of it is injected into the input element. Immediately thereafter, water (for laboratory use, ISO 3696:1987) is added to the hydrodynamic power source. Subsequently, the mixture is incubated for 2 min in the incubation chamber of the input element, this remaining incubation time being controlled by the combined timing/pressure element. The mixture is then transported to the bound/free element by the combined action of the pressure element and the hydrodynamic power source. After the bound/free separation process is completed, the latex solid phase is carried autonomously by the liquid flow to the output element. Here, the substrate components dissolve and, in the presence of the bound conjugate molecules, participate in the enzyme–substrate reaction; the color formed is observed by eye. The timing carried out by the circuit can easily be changed to 5 min, in which case the incubation outside of the circuit is not needed. The single bound/free element is operated by adding the assay mix to the element, followed immediately by addition of the wash liquid (water for laboratory use, ISO 3696:1987). At first, the liquid is carried through the device by capillary action and later, after wetting, by gravity. Results performance of individual fluid elements and reagents Bound/free element. The liquid front of the assay mix entering the element travels ∼10-15 mm in the channel beyond the start of the membrane (typical flux in these experiments, 0.5 μL/s), which corresponds to 0.5–1.5 s before the membrane and absorber are wetted and the aspiration power of the absorber becomes available; after this time, the liquid front comes to a halt. Without a latex solid phase in the assay mix, this position is ∼5 ± 1 mm from the start of the membrane; presumably, the particles increase the hydrodynamic resistance of the membrane by blocking a number of pores. The latex recovery result is denoted in Table 1 . If the membrane was not made hydrophilic, latex recovery substantially decreased. Including salt in the wash liquid also worsened the latex recovery, as did a decrease in the amount of latex solid phase initially applied in the assay mix. We determined the amount of unbound conjugate in the aliquots exiting the element and plotted the log-transformed values as a function of the time at which the aliquots exited the element (Fig. 6 , curve A). The data points show a linear decrease as function of time, as predicted by Eq. 5. By applying an estimated value for Dc, the diffusion coefficient of the conjugate molecules in serum, we could calculate the thickness of the residual layer (δ) from the value of the slope of curve A. Extrapolating the data of Tyn and Gusak (5) gave an estimated Dc of 1.8 × 10−11 • m2 • s−1 and a calculated δ of 90 μm. However, the layer thickness of the residual layer is not equal at all positions. In particular, the fact that the channel has a cross-section of 2 × 0.1 mm makes it likely that the residual layer at the corners is much thicker than in the middle part of the channel. Therefore, the calculated value of the layer thickness will be an average, depending on the size of the measured time window. Moreover, conjugate molecules located in the membrane pores and in the absorber can diffuse back into the channel, also influencing the calculated value of δ. Decreasing the surface area mutually used by the assay mix and the wash liquid decreased substantially the amount of unbound conjugate molecules finally leaving the element (Fig. 6 , curves B and C). For comparison reasons, we include the tabulated separation efficiency of the bound/free element in Table 1 . Track-etched membranes, having a smooth surface, provided the best results. When we used tortuous membranes, which have many dead spaces and a relatively rough surface, the wash efficiency dropped by 10-fold (results not shown). Pressure element. Measurement of the pressure necessary to displace a water or serum sample in a 2-mm-diameter Lexan chamber showed it to be <0.100 kPa. The maximum pressure buildup produced by filling the absorber with water was measured as 6.500 (±10%) kPa. Because the pressure to drive the displacement is much greater than the pressure barrier, one can apply Eq. 2 as a good approximation for determining the liquid flux of the liquid sample being displaced. The advancing contact angle of water on Lexan was found to be 85° ± 2°. The receding contact angle was difficult to measure; therefore, we used Eq. 6 to derive the receding contact angle: 61°. From these values we could predict the pressure barriers (Pbar) in various geometries. In principle, Eq. 7 can be used to calculate the receding contact angle of water in the absorber. However, during filling of the absorber with water, components of the absorber dissolve and thereby influence the surface tension value. The value of the pore sizes, as determined with the porometer, can also be influenced by this effect. Because no separation process is conducted with this element, the membrane component is omitted. In this case, the retreat effect (described above in Liquid transport by the element) can be easily observed. The water progresses into the channel to a position 5 ± 1 mm past the starting point of the absorber. After this, the absorber is wetted and the full aspiration power (unrestricted by any membrane) is available, such that the liquid front in the channel retreats to a position 1 ± 0.5 mm from the start of the absorber. Timing element. The time to fill an element was measured in a small circuit of a hydrodynamic power source connected in series to a combined timing/pressure element (see Fig. 4 ). The hydrodynamic power source included a vertical cylinder (where a water column introduces a hydrostatic pressure) and a membrane restriction. The delay time (δT) for the element was measured as ∼70 s. To calculate δT with the help of Eqs. 2–4, one must know the hydrodynamic flow resistance (Rcir) of the circuit in front of the element. This resistance is mainly determined by the membrane restriction, and the resistance of the channels connecting the elements can be neglected. The hydrodynamic resistance of the membrane restriction can be directly derived from the transmembrane flux and the surface area of the membrane (3.9 mm2). Furthermore, as mentioned above, the liquid retreats during the filling of the absorber; hence, the receding contact angle has to be used in Eq. 2. The values of constants and variables used in these calculations are listed in Table 2 . Note that Eq. 2 is valid for a slit where D ≪ W. In the experimental setup, D = W/20 and the different surfaces making up the channel can distort the meniscus. Given that one wall of the channel is the surface of the absorber and three walls are Lexan, we used estimates of the average advancing and receding contact angles. The resulting calculated δT was between 52 and 74 s, which is comparable with the experimentally measured value. The δT in the liquid flow introduced by elements can be used to control the incubation time of an ELISA performed as shown in Fig. 5 . Output element. As perceived by eye, the amount of free HRP still producing a color in the element within 5 min was 4 pg. Reagents. Testing the reagents independently of the fluid elements and circuits (see Materials and Methods) yielded a detection limit for HBsAg of 1 IU/mL. The test samples used were calibrated against standards provided by the Paul Ehrlich Institute, Langen, Germany. The dose–response curve is depicted in Fig. 7 . performance of fluid element circuits The detection circuit (Fig. 3 ) is a combination of a bound/free element and an output element. The detection limit was monitored and is tabulated in Table 1 . In this case the liquids are added in the same way as to a single bound/free element, and color formation is observed by eye. The integrated circuit contains several elements and is capable of carrying out an ELISA almost fully autonomously (Fig. 5 ). The circuit pictured uses an adapted bound/free element in which the assay mix (containing the sample) and wash liquid enter through separate channels, minimizing the area of mutually used surface. The resulting separation efficiency exceeds that of a bound/free element with only one entrance (see Table 1 ). Also, the detection limit is improved, probably because of the increased separation efficiency. Because the liquid sample and wash liquid are not in physical contact with each other in the pressure element, the wash liquid can still be used in the bound/free element. Also, because the wash liquid is released by the pressure element only after the liquid sample is deposited in the bound/free element, no extra timing for transport of the wash liquid has to be built in. Discussion The detection limit for material added to normal human serum, as detected with the integrated circuit, was similar to that for the Hepanostika kit for screening blood for HBsAg. The screening of clinical samples, including sero-conversion samples, with the integrated circuits is the subject of further study. In any event, the number of handling steps is much smaller than in the blood screening assay, and the total test time with the integrated circuit is fivefold less (Table 1 ). The larger surface-to-volume ratio of the dispersed solid phase, in comparison with a microtiter well as solid phase, probably accounts for this shorter test time with an almost equal detection limit. Also, the disperse nature of the solid phase decreases the diffusion distances during incubation of the assay mix. Remarkably, the detection limit of the integrated circuit is even better than that of the reagents tested independently of the circuit. Possibly the separation efficiency is higher than when testing the reagents separately. Unfortunately, separation efficiencies >99.9988% could not be determined accurately, so no definite conclusions can be drawn with respect to this. The detection circuit is generic in the sense that different analytes can be detected with the circuit. The integrated circuit can be used in several ways. When the assay mix is added to the circuit, the circuit is generic. When the reagents are self-contained in the chamber of the input element, the circuit is dedicated to one type of analyte. When different reagents for various analytes are stored in the chamber, multianalyte testing is possible. Alternative circuits for carrying out an ELISA are also possible, one theoretical example being shown in Fig. 8 . Additional types of analytical tests can also be carried out but are beyond the scope of this report. The demonstrated feasibility of the concept makes it possible to further develop manual tests based on fluid elements for application in less-sophisticated laboratories or in point-of-care situations. The small size of the assay components, the few handling steps, and simple operation could make the test accessible to nontechnical personnel; even the use at home by patients could be considered. Existing manual tests cannot offer a detection limit comparable with that of current blood screening assays in combination with a short test time and few handling steps. Future developments in instrument design could benefit from the concept. The complexity of conducting an ELISA is almost fully controlled by the circuit of fluid elements. If such a circuit of elements could be considered as a disposable “cuvette,” it could be used in combination with an instrument of relatively simple design—i.e., an instrument that would add the sample to the cuvettes and would read the results by optical or electrochemical means. The complex control software and complex mechanical robotics typical for current immunoanalyzers could be omitted and thereby decrease the need for servicing. The size of the instrument could decrease substantially and perhaps even utilize a different design: parallel processing of cuvettes. This might open a product line wherein the customer could determine the capacity of the instrument used by simply changing the number of slots for positioning such cuvettes in the instrument. In conclusion, the fluid element as an automation concept has some potentially very beneficial features: flexible design, low detection limit, and rapid, autonomous operation. The concept of creating circuits by combining fluid elements makes it likely that flexible different designs can be set up. Assay detection limits, as determined with analyte-supplemented material, are comparable with those for state-of-the-art blood screening assays—a necessary feature if fluid element assays are to be used for screening blood. The detection limit for clinical samples was not determined but is the subject of further study. Rapid test times, especially convenient in point-of-care testing and in an emergency situation, of 20 min can be obtained for the integrated circuit vs 90 min for a typical state-of-the-art blood screening assay. Moreover, because a circuit of elements can perform a test almost fully autonomously, without moving parts and without an external power source, one would expect a test system using these circuits to be highly reliable. We think that the features of the fluid element circuit concept—i.e., no moving parts and only two interfaces to the outside world (input and output)—could combine very well with the miniaturization technology currently used for chip fabrication and could yield an even more exciting future involving new diagnostic formats. Organon Teknika, Boseind 15, 5280 AB Boxtel, The Netherlands. Figure 1. Open in new tabDownload slide A schematic representation of the fluid element at the moment when the absorber is wetted. The movement of the liquid front in the channel is determined by the aspiration power of the absorber in combination with the contact angle (θ). At low aspiration power (Q2<Q1), the liquid front continues to advance in the channel while θ = θadv (the advancing contact angle). At medium aspiration power (Q2 = Q1), the liquid front comes to a halt while θ is in between θadv and θrec (θrec < θ < θadv). At large aspiration power (Q2 > Q1), the liquid front retreats while θ = θrec (the receding contact angle). Figure 1. Open in new tabDownload slide A schematic representation of the fluid element at the moment when the absorber is wetted. The movement of the liquid front in the channel is determined by the aspiration power of the absorber in combination with the contact angle (θ). At low aspiration power (Q2<Q1), the liquid front continues to advance in the channel while θ = θadv (the advancing contact angle). At medium aspiration power (Q2 = Q1), the liquid front comes to a halt while θ is in between θadv and θrec (θrec < θ < θadv). At large aspiration power (Q2 > Q1), the liquid front retreats while θ = θrec (the receding contact angle). Figure 2. Open in new tabDownload slide Left: a bound/free element (middle); a combination of a bound/free element and an output element, called a detection circuit (top); and an older version of the detection circuit (bottom); right: integrated circuits. In the top and middle devices (left panel), the membranes are heat-sealed onto the channel; the plastic parts are also connected by heat seals. In the older detection circuit (bottom device), the membrane and parts are connected by double-sided adhesive tape but the function is identical to that of the topmost device. In the integrated circuits in the right panel, the membrane is heat-sealed to one of the plastic parts, and the plastic parts are connected by nuts and bolts. Figure 2. Open in new tabDownload slide Left: a bound/free element (middle); a combination of a bound/free element and an output element, called a detection circuit (top); and an older version of the detection circuit (bottom); right: integrated circuits. In the top and middle devices (left panel), the membranes are heat-sealed onto the channel; the plastic parts are also connected by heat seals. In the older detection circuit (bottom device), the membrane and parts are connected by double-sided adhesive tape but the function is identical to that of the topmost device. In the integrated circuits in the right panel, the membrane is heat-sealed to one of the plastic parts, and the plastic parts are connected by nuts and bolts. Figure 3. Open in new tabDownload slide The bound/free element, the pressure element, the output element, the modified bound/free element of the integrated circuit, and the detection circuit (combination of a bound/free element and an output element). The modified bound/free element has separate entrances for sample liquid (the assay mix containing the sample) and wash liquid. The surface of the air vent of the output element is made hydrophobic to prevent leakage of liquid but to allow air to escape during filling with liquid. Except for the side view of the modified bound/free element of the integrated circuit, all diagrams are at the scale shown. Figure 3. Open in new tabDownload slide The bound/free element, the pressure element, the output element, the modified bound/free element of the integrated circuit, and the detection circuit (combination of a bound/free element and an output element). The modified bound/free element has separate entrances for sample liquid (the assay mix containing the sample) and wash liquid. The surface of the air vent of the output element is made hydrophobic to prevent leakage of liquid but to allow air to escape during filling with liquid. Except for the side view of the modified bound/free element of the integrated circuit, all diagrams are at the scale shown. Figure 4. Open in new tabDownload slide The timing circuit is a combination of a hydrodynamic power source and a combined timing/pressure element; it is used to test the timing element. Figure 4. Open in new tabDownload slide The timing circuit is a combination of a hydrodynamic power source and a combined timing/pressure element; it is used to test the timing element. Figure 5. Open in new tabDownload slide A schematic representation of the operation of the integrated circuit, consisting of one combined timing/pressure element and one modified bound/free element. For ease of viewing, the circuit is unfolded into one plane; in the actual device, the channels and elements are located in two levels. In addition to the fluid elements, an input element, a hydrodynamic power source, and an output element are used. The input element consists of a chamber into which the assay mix is deposited. Alternatively, this chamber can contain dry reagents, which are resuspended during injection of the sample. Figure 5. Open in new tabDownload slide A schematic representation of the operation of the integrated circuit, consisting of one combined timing/pressure element and one modified bound/free element. For ease of viewing, the circuit is unfolded into one plane; in the actual device, the channels and elements are located in two levels. In addition to the fluid elements, an input element, a hydrodynamic power source, and an output element are used. The input element consists of a chamber into which the assay mix is deposited. Alternatively, this chamber can contain dry reagents, which are resuspended during injection of the sample. Table 1. Physical parameters and assay performance of various formats. . Test time, min . Separation efficiency1 Latex recovery . . HBsAg detection limit, IU/mL . . . % . . . Control reagents 45 >99.9988 70–80 1 Bound/free element 15 99.9936± 0.0017 70–80 n.a. Detection circuit 15 n.m. n.m. 5b Integrated circuit 20 >99.9988 70–80 0.32 Hepanostika- Uni-form II3 90 n.m. n.a. 0.2 . Test time, min . Separation efficiency1 Latex recovery . . HBsAg detection limit, IU/mL . . . % . . . Control reagents 45 >99.9988 70–80 1 Bound/free element 15 99.9936± 0.0017 70–80 n.a. Detection circuit 15 n.m. n.m. 5b Integrated circuit 20 >99.9988 70–80 0.32 Hepanostika- Uni-form II3 90 n.m. n.a. 0.2 1 The percentage of unbound conjugate molecules not recovered at the exit of the bound/free element. Efficiencies >99.9988% could not be determined accurately and are simply indicated with >. 2 The concentration of analyte where the eye observes a color formation significantly different from that for a blank sample. 3 HBsAg blood-screening assay of Organon Teknika included for comparison. n.m., not measured; n.a., not applicable. Table 1. Physical parameters and assay performance of various formats. . Test time, min . Separation efficiency1 Latex recovery . . HBsAg detection limit, IU/mL . . . % . . . Control reagents 45 >99.9988 70–80 1 Bound/free element 15 99.9936± 0.0017 70–80 n.a. Detection circuit 15 n.m. n.m. 5b Integrated circuit 20 >99.9988 70–80 0.32 Hepanostika- Uni-form II3 90 n.m. n.a. 0.2 . Test time, min . Separation efficiency1 Latex recovery . . HBsAg detection limit, IU/mL . . . % . . . Control reagents 45 >99.9988 70–80 1 Bound/free element 15 99.9936± 0.0017 70–80 n.a. Detection circuit 15 n.m. n.m. 5b Integrated circuit 20 >99.9988 70–80 0.32 Hepanostika- Uni-form II3 90 n.m. n.a. 0.2 1 The percentage of unbound conjugate molecules not recovered at the exit of the bound/free element. Efficiencies >99.9988% could not be determined accurately and are simply indicated with >. 2 The concentration of analyte where the eye observes a color formation significantly different from that for a blank sample. 3 HBsAg blood-screening assay of Organon Teknika included for comparison. n.m., not measured; n.a., not applicable. Figure 6. Open in new tabDownload slide Residual fractions of conjugate molecules in the wash liquid as function of the elution time. Curves A (•) show the results obtained with the bound/free element depicted in Fig. 3 . In this case, the distance between entrance and the liquid front in the channel during filling of the absorber is ∼25 mm. Curves B (▪) and C (▴) show results for when this distance is 5 and <0.5 mm, respectively. For this study, (B and C) the position of the liquid front was controlled by a pump that added liquid through an extra hole at the position of the membrane. Each line refers to a single experiment. Figure 6. Open in new tabDownload slide Residual fractions of conjugate molecules in the wash liquid as function of the elution time. Curves A (•) show the results obtained with the bound/free element depicted in Fig. 3 . In this case, the distance between entrance and the liquid front in the channel during filling of the absorber is ∼25 mm. Curves B (▪) and C (▴) show results for when this distance is 5 and <0.5 mm, respectively. For this study, (B and C) the position of the liquid front was controlled by a pump that added liquid through an extra hole at the position of the membrane. Each line refers to a single experiment. Table 2. Values of constants and variables at 25 °C. Earth properties . Water properties . Interaction parameters . g = 9.8 m s−1 ρ = 1.0 × 103 kg m−3 θadv = 30–85 (Eq. 4) γ = 7.2 × 10−2 kg s−2 θ = θrec = 30–60 (Eq. 2) μ = 8.9 × 10−4 kg m−1s−1 heq = 1 × 10−3 m Dimensions of element Dimensions circuit Absorber properties D = 1 × 10−4 m z = 6 × 10−2 m Vabs = 9.52 × 10−8 m3 W = 2 × 10−3 m Rcir = 1.03 × 1012 kg m−4s−1 Por = 0.943 (compressed) hele = 1.3 × 10−2 m Earth properties . Water properties . Interaction parameters . g = 9.8 m s−1 ρ = 1.0 × 103 kg m−3 θadv = 30–85 (Eq. 4) γ = 7.2 × 10−2 kg s−2 θ = θrec = 30–60 (Eq. 2) μ = 8.9 × 10−4 kg m−1s−1 heq = 1 × 10−3 m Dimensions of element Dimensions circuit Absorber properties D = 1 × 10−4 m z = 6 × 10−2 m Vabs = 9.52 × 10−8 m3 W = 2 × 10−3 m Rcir = 1.03 × 1012 kg m−4s−1 Por = 0.943 (compressed) hele = 1.3 × 10−2 m Table 2. Values of constants and variables at 25 °C. Earth properties . Water properties . Interaction parameters . g = 9.8 m s−1 ρ = 1.0 × 103 kg m−3 θadv = 30–85 (Eq. 4) γ = 7.2 × 10−2 kg s−2 θ = θrec = 30–60 (Eq. 2) μ = 8.9 × 10−4 kg m−1s−1 heq = 1 × 10−3 m Dimensions of element Dimensions circuit Absorber properties D = 1 × 10−4 m z = 6 × 10−2 m Vabs = 9.52 × 10−8 m3 W = 2 × 10−3 m Rcir = 1.03 × 1012 kg m−4s−1 Por = 0.943 (compressed) hele = 1.3 × 10−2 m Earth properties . Water properties . Interaction parameters . g = 9.8 m s−1 ρ = 1.0 × 103 kg m−3 θadv = 30–85 (Eq. 4) γ = 7.2 × 10−2 kg s−2 θ = θrec = 30–60 (Eq. 2) μ = 8.9 × 10−4 kg m−1s−1 heq = 1 × 10−3 m Dimensions of element Dimensions circuit Absorber properties D = 1 × 10−4 m z = 6 × 10−2 m Vabs = 9.52 × 10−8 m3 W = 2 × 10−3 m Rcir = 1.03 × 1012 kg m−4s−1 Por = 0.943 (compressed) hele = 1.3 × 10−2 m Figure 7. Open in new tabDownload slide Dose–response curve of the HBsAg assay tested independently of the fluid elements and circuits (see Materials and Methods). Figure 7. Open in new tabDownload slide Dose–response curve of the HBsAg assay tested independently of the fluid elements and circuits (see Materials and Methods). Figure 8. Open in new tabDownload slide An alternative circuit for conducting an ELISA, based on the use of a combined timer/pressure element and a combined bound/free + pressure element. In this case, the output element is used only for storing the impregnated plates and resuspending the substrate components. The reaction producing visible results is executed in the combined bound/free + pressure element. Figure 8. Open in new tabDownload slide An alternative circuit for conducting an ELISA, based on the use of a combined timer/pressure element and a combined bound/free + pressure element. In this case, the output element is used only for storing the impregnated plates and resuspending the substrate components. The reaction producing visible results is executed in the combined bound/free + pressure element. We acknowledge B. Krutzer and S. Vos for supplying the reagents and carrying out experiments with the detection circuit; R. Hoeben and H. van der Linden for technical support; T. Beumer for sharing his knowledge about wash processes and for support in conducting several of the fundamental experiments; and W. Carpay for stimulating discussions about fundamentals and methods. 1 Skeggs L. An automatic method for colorimetric analysis. Am J Clin Pathol 1957 ; 28 : 311 -322. Crossref Search ADS PubMed 2 Ruzicka J. Flow injection analysis, from test tube to integrated microconduits. Anal Chem 1983 ; 55 : 1040A -1053A. 3 Hiemenz PC. Principles of colloid and surface chemistry, 2nd ed. New York: Marcel Dekker, 1986:338pp.. 4 Beumer T, Stoffelen E, Smits J, Carpay W. Microplate washing: process description and improvements. J Immunol Methods 1992 ; 154 : 77 -87. Crossref Search ADS PubMed 5 Tyn MT, Gusak TW. Prediction of diffusion coefficients of proteins. Biotechnol Bioeng 1990 ; 35 : 327 -338. Crossref Search ADS PubMed © 1997 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Fluid elements—a concept for automation of diagnostic tests
JF - Clinical Chemistry
DO - 10.1093/clinchem/43.2.369
DA - 1997-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/fluid-elements-a-concept-for-automation-of-diagnostic-tests-4O74Op3EDg
SP - 369
EP - 378
VL - 43
IS - 2
DP - DeepDyve
ER -