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Keywords:

  • liquid marbles;
  • micro-bioreactors;
  • diagnostic assays;
  • superhydrophobicity;
  • blood typing

Due to their unique properties, liquid marbles have been the subject of a collection of studies in the past decade, centered on fundamental research on their properties, as well as their practical applications.1–37 These liquid droplets, enwrapped with solid powder while having no direct contact with the supporting substrate, may be exploited for a wide range of applications ranging from, but not limited to, the displacement of a small volume of liquid without any leak left behind,2 water surface pollution detection,13 gas detection, gas–liquid reactions,8, 30, 31 and, last but not the least, preparation of microreactors.7, 30, 31, 34 With numerous powder types available, the fabrication options of liquid marbles seem to be infinite. This enables the design of tailor-made liquid-marble-based systems for intended applications.

To our knowledge, fabrication of microreactors by forming liquid marbles for the purpose of containing chemical reactions at the micrometer scale has been proposed by only a limited number of studies. For instance, Xue et al.34 have shown that a liquid marble coated with magnetic powder can be used as a miniature chemical reactor to either encapsulate the reagents in a single marble, or in two separate marbles, which could coalesced afterwards to trigger the reaction. The authors used fluorinated decyl polyhedral oligomeric silsesquioxane and magnetic powder aggregates to generate stable liquid marbles capable of encapsulating liquids of either high or low surface tension. They also demonstrated a chemiluminescence reaction between hydrogen peroxide and bis (2,4,6-trichlorophenyl) oxalate and a dye to prove the concept of the controllable liquid marble micro-reactors.

Tian et al.30, 31 showed that the porous nature of the liquid-marble shell could be used to allow gases to transport through the marble shell. They demonstrated the use of liquid marbles formed with gas-reactive indicator solutions to detect gases. Bormashenko et al.8 have also reported the use of polyvinylidene fluorid particles of micrometer size for fabrication of a liquid marble microreactor containing ammonia acetate, acetic acid, and acetylacetone, which is then exposed to formaldehyde vapor to trigger the reaction.

In this study we present the use of liquid marble as micro-bioreactors for biological reactions and diagnostic assays. We choose human blood grouping (ABO and Rh) as the biological system to demonstrate the use of liquid marble as a micro-bioreactor in practical diagnosis involving human blood, which is the most biologically informative human body fluid. The significant advantages of liquid marble micro-bioreactor are: First, it requires relatively small amount of samples and reagents. Second, it reduces biohazards, since the power-wrapped biological sample makes no contact to the surface of the supporting substrate. Third, the control of the bioreactions can be made by either coalescing marbles containing different reagents or by injecting into the marble of different reagents. Fourth, marbles are low in cost and therefore disposable. The construction of the micro-bioreactors for this work is simple; in each blood grouping test three drops (3 × 10 μL) of a blood sample were used to prepare three “blood marbles”. Three antibody solutions (Anti-A, Anti-B and Anti-D) were injected into the three blood marble to initiate the test. Subsequently, ABO and Rh blood grouping is studied by monitoring whether or not haemagglutination reaction occurs inside each of the blood marble micro-bioreactor.

The presence or absence of certain antigens on the surface of a red blood cell (RBC) is an intrinsic biological property which determines a person's blood group. On the other hand, antibodies existing in the blood plasma are available to protect the body when threatened by hostile antigens. According to Landsteiner's Law, when an RBC possesses certain antigens on its surface, the corresponding antibody is absent in the blood plasma and vice versa.38 Blood grouping is a basic yet essential test to be performed prior to a blood transfusion to avoid the consequences of incompatibility, which may lead to a fatal haemolytic reaction. A few examples of the current techniques of blood grouping include dry instant blood typing plate.39 Microplate-based techniques,40–42 and integrated microfluidic biochips are among other options of blood grouping.43 Recently, interesting progress has been made in the fabrication of low-cost, disposable and easy to use paper-based44 and thread-based45 blood typing devices. The liquid marble micro-bioreactor method we report herein has the same advantages in terms of, low-cost, disposability, not relying on any medical facilities.

The microreactors in this work were made by coating blood drops with hydrophobic powder of precipitated calcium carbonate (PCC). An antibody solution was subsequently injected into the micro-bioreactors to test for haemagglutination. Scheme 1 shows the schematic illustration of the steps of the experiment. Before the antibody injection, all blood marbles were in a homogeneous red color. Immediately after the inection of antibody solution into the blood marbles, strong darkening of the marbles injected with Anti-A was observed. This is because commercial Anti-A solution is color-coded with a blue dye for identification purpose (Figure 1). If haemagglutination reaction occurs, the initial uniform red color of the blood marble separates into two clearly discernible parts of light- and dark-red colors due to the precipitation of the agglutinated RBCs to the bottom of the marble. The appearance of such color separation of a blood marble signals the agglutination reaction, indicating the presence of the corresponding antigen on the surface of RBCs. On the other hand, if color separation of the blood marble does not occur, it indicates that the corresponding antigens are absent. The blood grouping results of A+, B+, O+ and O– samples can be seen in (Figure 1). It is worth noting that due to the strong red color of the blood samples, if the haemagglutination does not occur, even by providing a strong backlighting, the blood marble micro-bioreactor will still remains uniformly dark-red in color and opaque. On the other hand, if haemagglutination reaction occurs, a lighter-colored upper part develops when agglutinated RBCs settles to the lower part of the marble.

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Scheme 1. The Schematic illustration of the steps of microreactor preparation and blood-type identification. A) Blood (10 μL) is placed on a hydrophobic PCC powder bed to form the blood marble. B) An antibody solution (10 μL) is injected inside the blood marble to complete the preparation of the microreactor. C) When the corresponding antigens are not present on the surface of RBCs, no separation is visible. D) When the corresponding antigens are present, RBC agglutination reaction will take place; this will result in the separation of marble color into two distinct light (top) and dark (bottom) parts.

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Figure 1. Summary of blood typing results after the corresponding antibodies are injected into the marble microreactor (20 μL). Green ticks are added to the photos where the separation of agglutinated RBC is observed. The cross signs are added to photos where agglutination caused color separation is not observed.

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This method requires three blood marble micro-bioreactors to determine the blood group (ABO RhD) of a blood sample. The interpretation of the blood grouping test result can be made following this example: For a blood marble made of a B+ sample, injections of Anti-B and Anti-D solutions will cause the separation of uniformly red-colored marbles into a light-red (top) and dark-red (bottom) parts. However, the injection of Anti-A will not change the color uniformity of the marble, as no haemagglutination will occur between a B+ sample and anti-A (Figure 1).

According to the literature, blood surface tension is lower than that of water.46 This might explain the higher than usual deformation of the blood marbles, compared with the pure water marble. Further study of the physical properties of blood marbles can be done in a our future investigations. Nevertheless, based on our observations, blood marble micro-bioreactor has the potential to be used rapid blood typing tests. Only a few seconds of gentle shaking of the marble containing the blood and antibody mixture is enough to initiate the haemagglutination reaction. Whilst the main powder used for this study was PCC treated with stearic acid,47 we have also used PTFE powder (100 μm particle size) (Figure 2) just to demonstrate the feasibility of using other powders for the same application. The two powders used in this work are just examples of the numerous powder type options that can be used for the same purpose.

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Figure 2. A) A blood marble (20 μL) micro-bioreactor made of A+ blood and hydrophobic PCC powder after the injection of Anti-A solution. B) A blood marble (20 μL) micro-bioreactor made of A+ blood and PTFE powder after the injection of Anti-A solution.

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The choice of PCC was made mainly because of its low cost, availability, environmental compatibility, and ease of hydrophobization. PCC crystallites are about 1 μm in length but clusters containing a few crystallites can have large sizes of several micrometers. Further aggregations of the clusters can be seen on the surface of the blood marble. However, the color change caused by haemagglutination is clearly visible. Since only a minute amount of powder is needed to form a marble, and considering the low-cost of the PCC powder, this method can be regarded as one of the most inexpensive methods suitable for ABO and Rh blood typing. Furthermore, after the blood typing test, the used marble microreactor can be burnt to eliminate any potential biohazards. We believe that this study may open a new door for the further biological applications of using liquid marble as micro-bioreactors.

Experimental Section

  1. Top of page
  2. Experimental Section
  3. Acknowledgements

Four blood samples of known types were acquired and stored in Vacutainer® test tubes containing lithium-heparin anticoagulant from a pathological laboratory. Precipitated calcium carbonate powder (Precarb 100, BASF, which was then treated in-house with stearic acid) was used as the coating powder.46 To prepare a blood marble, a drop of blood was placed on the hydrophobic PCC powder bed inside a Petri dish using a micropipette. The Petri dish was then shaken gently to allow the PCC particles to cover the blood drop uniformly. The same method was used to prepare PTFE coated marbles; a contact angle measurement system (Dataphysics OCA230, Germany) was used to take images of both marbles after haemaglutination inside the marbles has occurred. Epiclone Anti-A (color-coded blue), Anti-B (color-coded yellow) and Anti-D (colorless) monoclonal grouping reagents were acquired commercially from Commonwealth Serum Laboratory, Australia. The same volume of an as-received antibody was then injected into a blood marble using the micropipette, which finally constructed the micro-bioreactor for the RBC agglutination reaction to take place. This procedure was repeated three times to prepare three microreactors containing the same blood sample, but different antibodies. The blood type was then determined based on the method described above. Photos were taken using a Mju 9010 Olympus digital camera. A spatula was used to transfer the microreactor onto a microscope glass slide and a suitable lighting condition was provided with a light source (Microlight 150, Fibreoptic lightguides, Australia).

Acknowledgements

  1. Top of page
  2. Experimental Section
  3. Acknowledgements

The authors would like to acknowledge Dr. Mohammad al-Tamimi for kindly providing the blood samples. Monash University postgraduate scholarships, as well as funding received from ARC LP0989823, are gratefully acknowledged.