Patent Application
20150079617
The invention relates to a method for determining a set of biochemical parameters in body fluids such as blood, blood serum or blood plasma. The invention comprises also the use of specially selected pairs of materials and liquids for determining biochemical parameters of a body fluid.
The state of the art knows many diagnostic methods in this study referred to as biochemical methods aiming at quantitative measurement (determination) of biochemical parameters of blood. At present, known groups of such assays of biochemical parameters include: assays of substrates, enzymes, electrolytes, specific proteins, monitoring of concentrations of drugs and intoxicants, concentrations of hormones, cancer markers, cytokines and other types of proteins, as well as all other parameters that can be determined using photometric methods. These assays can be performed in various materials of human or animal (veterinary) origin, including: whole blood, serum, plasma, cerebrospinal fluid, urine or other fluids from body cavities. These tests are usually performed in analytical laboratories by laboratory diagnosticians or medical testing technicians (material taken from humans) or in veterinary practices by trained personnel (material from animals). More and more often, however, such testing is performed as a part of a point-of-care or individual diagnostics outside the analytical laboratory, in emergency stations, intensive care units, specialised ambulances, directly by physicians, paramedics, nurses or other trained personnel.
For obvious reasons, the equipment used for individual or point-of-care assays should be easily portable, capable of providing test results within single minutes, and first of all such equipment must be reliable. In this context reliability shall mean the metrological reliability, i.e., the level of accuracy and reproducibility of the results obtained with a given instrument.
The development of microfluidics has enabled fabrication of a series of small, portable constructions that might be applied in medical diagnostics, especially in the area of individual or point-of-care diagnostics [e.g., A. Arora, G. Simone, G. B. Salieb-Beugelaar, J. T. Kim, A. Manz, Analytical Chemistry 82, 4830 (2010)]. In the underlying assumptions, these constructions are capable of making use of droplet flows [e.g., A. B. Theberge, F. Courtois, Y. Schaerli, M. Fischlechner, C. Abell, F. Hollfelder, W. T. S. Huck, Angewandte Chemie International Edition, 49, 5846 (2010)], i.e., such ones where two immiscible phases, such as water and oil, are introduced into a microfluidic system. In analogy to emulsification processes, the phase used to form droplets is referred to as the dispersed phase, and the phase in which the droplets are suspended is referred to as the continuous (dispersing) phase. The droplets (or bubbles), surrounded by another fluid spontaneously assume a spherical shape; when squeezed by channel walls they assume a shape of flattened ellipsoids or discs. It has been shown already that such systems are suitable for determining both single biochemical parameters and sets of multiple biochemical parameters using a single device.
It is a necessary prerequisite for formation of and control over a droplet flow that the dispersed phase does not wet the walls of the system (channels), as opposed to the continuous phase that must superbly wet the walls. Since biochemical assays use a very broad variety of reagents, a very important technical issue is to select the polymer used to fabricate the microfluidic system and the continuous liquid so, that no reagent, or at most possibly a few reagents only wet the channel walls in the presence of the continuous liquid. Likely, the assayed material (e.g., human or animal blood serum) must not wet the channel walls in the presence of the continuous liquid.
The Authors of the present invention have tested a very broad set of reagents for biochemical blood testing and a broad range of polymers and continuous liquids, and found unexpectedly preferred combination of polymer and continuous liquid that allow performing biochemical assays inside droplets formed and residing in the microfluidic systems, i.e., in microchannels inside the microfluidic cartridges.
In particular, the Authors of the present invention have confirmed that particularly preferred combinations were composed of polymer materials commonly used in industry: polypropylene, polyethylene and cyclic olefin copolymer (COC). These materials performed very well in a combination with oil—hexadecane. The droplets formed on the surfaces of these materials in the presence of hexadecane had a large contact angle, and the parameter is of key importance in forming droplets in a system. Unexpectedly, satisfactory results were obtained for the combination of Teflon and Fluorinert.
According to the invention, the method for determining biochemical parameters of a body fluid, wherein a sample of said body fluid in the form of a droplet is transported through a channel of a microfluidic system using a carrier liquid, mixed with a reagent thus initiating a chemical reaction between the sample and the reagent, and the result of the chemical reaction is measured, preferably with a spectrophotometer, whereby the said biochemical parameters of the body fluid are determined, is characterised in that the material used for fabrication of the microfluidic system and the said carrier liquid is pair of Teflon and Fluorinert.
Preferably, the said reagent is selected from the group comprising: acp (acid phosphatase), alat (alanine aminotransferase), albumin, alp (alkaline phosphatase), alpha-fetoprotein, alpha-1-microglobulin, amylase, asat (aspartate transaminase), aso (anti-streptolysin O), bil direct (direct bilirubin), bil total (total bilirubin), calcium, ceruloplasmin, cholesterol, cholinesterase, ck (creatine kinase), ck MB (creatine kinase MB), complement C3, complement C4, crp (C-reactive protein), cystatin C, D-dimer D, ethanol, phenobarbital, ferrum, ferritin, fibrinogen, ggt (gamma-glutamyltransferase), glucose, haptoglobin, hbdh (α-hydroxybutyrate dehydrogenase), hdl cholesterol, HbA1C (haemoglobin), immunoglobulin A, immunoglobulin E, immunoglobulin M, carbamazepine, creatinine, alpha-1-acid glycoprotein, ldh (lactate dehydrogenase), Idl cholesterol, lipase, lipoprotein, Mg (magnesium), copper, myoglobin, lactates, paracetamol, phosphorus, potassium, rf (rheumatoid factor), salicylates, sodium, theophylline, tg (triglycerides), total protein, ua (uric acid), uibc (unsaturated iron binding capacity), urea, urine protein.
The invention comprises also the use of a pair of the material and the liquid—Teflon and Fluorinert—for determining biochemical parameters of a body fluid.
In a non-limiting embodiment, the static 3 and the dynamic 8 contact angles are determined, said angles formed by the serum 5 and the biochemical reagents 5 with the surface 1 of the polymer plate in the atmosphere of the selected continuous fluid 4. In a non-limiting example, the following continuous fluids 4 were tested: hexadecane, silicon oil with a viscosity of 20 cSt, paraffin oil, mineral oil, Fluorinert FC 3283, Fluorinert FC 40, Fluorinert HFE 7100. The following polymer substrates 1 were used in tests: Dyneon, Teflon, polydimethylsiloxane (PDMS), polystyrene, polyethylene, polypropylene (two types, in the following referred to as PP and PPR), styrene—prop-2-enonitrile copolymer (SAN), polystyrene GPPS and polycarbonate, cyclic olefin copolymer (two types, in the following referred to as COC 5013 and COC 6015).
The wetting of substrates 1 by reference normal (HN) and pathological (HP) serum 5 was tested. The HN/HP serum is produced on the basis of the human serum. It is used as a measurement control of concentrations of organic and inorganic components, and of the activity of enzymes. Most parameters tested in the HN serum are within the range of normal values for adults, whereas the parameters obtained for HP mostly differ from the values considered as normal. The dynamic contact angle was also studied for serums with various dilutions. The dilution was performed using physiological saline (0.9% sodium chloride).
Table 1 shows a list of biochemical assays and the volume ratio of reagents and serum used in the reaction (markings: S-serum, R1-reagent 1, R2-reagent 2). Depending on the parameter being determined, single-reagent (R1) or dual-reagent (R1 and R2) reagents were used. For most items, the Table shows English reagent abbreviations with full names given in parentheses.
In the following part of the description we refer to the measurements of the static angle 3—the angle formed between the polymer substrate 1 and the plane 2 tangent to the interface of the dispersed phase 5 and the continuous phase 4 under static conditions, on a horizontally placed substrate (
In the case of some materials (polypropylene, polyethylene), two plate types were used in tests. In the first case, the surface of the plate was roughened, i.e., the plate had a number of unevennesses (notches) on the surface. In this case, reagent droplets were dispensed perpendicularly to the unevennesses (notches) to check the effect of surface unevennesses on the dynamic contact angle. The second type of the plate surface was the even surface. In a non-limiting embodiment, even plates are fabricated by casting the polymer on a polished metal matrix (e.g., aluminium or steel one). In a non-limiting embodiment, manipulations with droplets on the polymer substrates mentioned in the present patent application can be made at room temperature.
To assure that the results are clearer, hereinafter we use the following markings coding the minimum inclination of the substrate 1, for which the droplet started to flow:
Where the dynamic angle 8 is not given in the tables below, then it means that for a given inclination 7 the droplet flew so fast that taking a picture was very difficult.
In a non-limiting embodiment, the following, not much preferable angles were measured for reagents deposited on a substrate made of PDMS in the atmosphere of a silicon oil with a viscosity of 20 cSt (HP—pathological serum; HN—normal serum; e.g., x2—it means that the serum was diluted twice;)
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of PDMS and surface-modified with Aquapel (waterproof silane-siloxane sealer) in the atmosphere of Fluorinert 3283 fluorinated oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of polypropylene (PPR) in the atmosphere hexadecane oil. The PPR substrate was roughened.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on an even substrate made of PPR in the atmosphere of hexadecane oil.
In a non-limiting embodiment the following contact angles were measured for reagents deposited on a substrate made of PPR in the atmosphere of mineral oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of PPR in the atmosphere of paraffin oil.
In a non-limiting embodiment, the following contact angles were measured for reagents, for which the results of the tests on a PPR substrate in hexadecane oil were unfavourable, on a polypropylene (PP) substrate in the atmosphere of the same oil (hexadecane).
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of PP in the atmosphere of paraffin oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a polyethylene substrate containing small unevennesses in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on roughened polyethylene substrate in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of polyethylene in the atmosphere of mineral oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of GPPS in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of GPPS polystyrene in the atmosphere of mineral oil.
In a non-limiting embodiment, the following contact angles were measured for serum (HN—normal control serum, and HP—pathological control serum) and serum dilutions deposited on substrate made of dedecylamine-modified polycarbonate in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of PS polystyrene in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of SAN polymer in the atmosphere of mineral oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of SAN polymer in the atmosphere of paraffin oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of Dyneon polymer in the atmosphere of Fluorinert FC-40 fluorinated oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of Dyneon polymer in the atmosphere of Fluorinert FC 3283 fluorinated oil:
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of Dyneon polymer in the atmosphere Fluorinert FC-7100 fluorinated oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of Teflon polymer in the atmosphere of Fluorinert THF 7100 fluorinated oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of Teflon polymer in the atmosphere of Fluorinert FC-3283 fluorinated oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of cyclic olefin copolymer (COC) 5013 in the atmosphere of hexadecane oil.
In a non-limiting embodiment, the following contact angles were measured for reagents deposited on a substrate made of cyclic olefin copolymer 6015 in the atmosphere of hexadecane oil.
Considering the conditions mentioned in the introduction, based on the measurements of contact angles, the Authors of the present invention have unexpectedly discovered that the most preferred combination of polymer and continuous liquid for performing biochemical assays in droplets manipulated inside microfluidic cartridges is Teflon and Fluorinert HFE-7100.
The Authors of the present invention have unexpectedly discovered that the combination of Teflon and Fluorinert HFE-7100 enable controlled formation of droplets of biochemical reagents (Table 2) and manipulating these droplets inside microchannels or microchambers in the microfluidic cartridges.
A microfluidic system has been fabricated from polypropylene. The scheme of the system is shown in
The microfluidic systems and the method for transporting microdroplets using carrier liquids (continuous liquids) in these systems are known in the state of the art, e.g., from a patent application WO2011/090396. Likely, the method for determining concentrations of, for instance, albumin, bilirubin or creatinine, and many other biochemical parameters in a sample using spectrophotometric analysis is known in the state of the art, whereas the selection of the material for fabrication of the microfluidic system, the carrier liquid and the reagent constitute the element of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| P-397071 | Nov 2011 | PL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/067861 | 9/12/2012 | WO | 00 | 11/25/2014 |
