The authors have declared that no competing interests exist.
Measurement of the height of a packed column of cells or beads, which can be direclty related to the number of cells or beads present in a chamber, is an important step in a number of diagnostic assays. For example, haematocrit measurements may rapidly identify anemia or polycthemia. Recently, user-friendly and cost-efficient Lab-on-a-Chip devices have been developed towards isolating and counting cell sub-populations for diagnostic purposes. In this work, we present a low-cost optical module for estimating the filling level of packed magnetic beads within a Lab-on-a-Chip device. The module is compatible with a previously introduced, disposable microfluidic chip for rapid determination of CD4+ cell counts. The device is a simple optical microscope module is manufactured by 3D printing. An objective lens directly interrogates the height of packed beads which are efficiently isolated on the finger-actuated chip. Optionally, an inexpensive, battery-powered Light Emitting Diode may project a shadow of the microfluidic chip at approximately 50-fold magnification onto a nearby surface. The reader is calibrated with the filling levels of known concentrations of paramagnetic beads within the finger actuated chip. Results in direct and projector mode are compared to measurements from a conventional, inverted white-light microscope. All three read-out methods indicate a maximum variation of 6.5% between methods.
Enumeration of cells, and particularly of specific cell sub-populations, constitutes an important step in diagnostic assays and also in life science research. Cell-counting technologies, based on flow cytometry, are well established and have been widely used in research and clinical laboratories for decades [
Metastatic cancer is recognised as the most serious stage of the disease and is responsible for approximately 90% of related deaths. Via the vascular or lymph systems, CTCs reach distal sites where they may seed metastasis [
Since its emergence in the early 1980s [
Diagnosis is the first stage of the treatment cascade, and is sometimes referred to as ‘aware of status’. This is followed by 1. link to medical care, 2. continuation of medical care, 3. ART, and 4. viral suppression. Yet, this first diagnostic step remains a major bottleneck for initiation of treatment in Sub-Saharan Africa; in 2012 only 51% of those living with HIV were aware of their status while, strikingly, in certain countries it is even as low as 10% [
The recent development of targeted ‘Point-of-Care’ (PoC) instrumentation, which is specifically designed to meet the ASSURED criteria for deployment in remote settings with poor health infrastructure, shows great potential to remove the ‘aware of status’ bottleneck [
The concentration of CD4-expressing T-helper cells (Th-cells) in the blood of a patient is considered a reliable measure of the status of HIV infection. While the number of CD4+ cells varies according to demographic, environmental and health factors [
Recently, Glynn
In this paper we present an optical reader which is compatible with the chip introduced by Glynn
(i) The chip filled with dyed water to highlight its geometry. (ii) Schematic of chip and (iii) chip operation. To operate, the chip is first primed with buffer through degas driven flow. To load, the P1 chamber is depressed and sample is pipetted into sample input port. The P1 chamber is then released and, as the chamber relaxes to its earlier shape, the sample id drawn through the chip and past the capture chamber. Repeated pressing and release of the chamber reciprocally pumps the sample through the separation chamber. Figure is reproduced from Glynn
In this study our reader measures height of bead conjugate in both ‘direct interrogation’ and ‘projector’ modes, and, for comparison,
The microfluidic chips are prototyped using the method previously described by Glynn
When cured, the PDMS is cut from the mould and a sample loading hole is defined using a dot punch. Following this, the PDMS slab is placed on a plasma-treated standard glass slide and bonded by stiction. The PDMS-glass hybrid was then secured, using pressure sensitive adhesive (PSA – Adhesives Research, Ireland), to a supporting bracket cut from polymethylmethacrylate (PMMA) (Radionics, Ireland) by a CO2 laser writer (Epilog Zing, USA).
Prior to use, the chips are kept under vacuum for at least 1 hour. To prime, a large drop of priming buffer composed of phosphate buffered saline (PBS) pH 7.4, 0.1% w/v bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetic acid (EDTA) covering the inlet for sample-loading was deposited on the surface of the PDMS. This buffer is drawn into the channels by degas flow [
As described above, the reader (
(i) The low-cost device is 3D-printed from four separate parts. Additional parts are a threaded screw, objective lens and diffusion plate. The finger actuated chip is inserted into a moveable chip holder. Its chip location relative to the objective lens, and thus the focus, can be finely adjusted by turning the focusing screw. (ii) The chip is interrogated by looking through the objective. The packed height of CD4+ cells and bead conjugate can be estimated from graduated hatch marks. Alternatively, the hatching can be calibrated as ‘treat’ or ‘no treat’ based on clinical guidelines. (iii) Image of the optical reader. Also in the image is an alternative lens holder for larger objectives. Additionally, a ‘Projector insert’, a powerful, low cost LED powered by a 3V battery, can be placed into the reader in place of the diffusion plate. In this case, the shadow of the packed cells and graduated hatch marks can be projected against a wall or floor.
Two identical chip-readers were manufactured for this study. One is configured for direct interrogation; for the second device operating in projector mode (
(i) Schematic of the reader in projector mode. A shadow of the read chamber can be projected onto a wall from a distance of ~1 m and can easily be discerned in a dim or dark room (ii) an image (acquired using a smartphone) of the read chamber shadow projected onto a wall. This is read as ‘5’ relative to the graduated markings. The projected image is approximately 50 times larger than the read chamber.
The reader was characterised by testing the system with non-fluorescent magnetic beads (CD14+ tagged beads, Dynabeads® T4 Quant Kit, Thermo-Fischer Scientific). The sample preparation methods used were previously described by Glynn
Bead packing is first measured using the ‘direct interrogation’ optical reader by the user conducting the experiments. The chip is then immediately transferred to the reader operating with shadow projection on the wall. In both cases, the height is estimated by the user by rounding the highest visible peak up to the next notch. The chips are then imaged using an Olympus IX81 inverted microscope (Olympus, Tokyo, Japan) by another experimenter. The user again measures the packed bead height using the scale bars integrated into the chip. A consistent rule is applied where the reading is rounded up from the highest point.
As can be seen in
We have presented an optical device that can be used for interrogating a previously published finger-actuated magnetophoretic chip. The reader works consistently in two different modes. In the first mode, bead counts can be obtained by direct ‘eye-balling’; in the other mode, a shadow of the measurement chamber is projected on to a nearby surface. Either method shows good agreement with measurements made by (blinded) experiments using a commercial microscope. In particular, in a darker environment, the projector proved to be user-friendly and the images are easy to interpret.
The system prototype costs approximately €60 in materials (approx. €30 for the optical lens, €5 for masonry bolt and projector electronics and approx. €25 for 3D printing material and machine time). However, towards mass production, it can be envisaged that the plastic components, and also the lenses, can be manufactured by cost-efficient injection moulding. Indeed, there is potential these components could be moulded as one contiguous part. Thus, we estimate that the cost of this fully re-usable instrument could be as low as €10.
As a next step, we intend to pair this reader with a low-cost smartphone [
This file contains the data acquired during experimentation.
(XLSX)
This work was supported in part by Science Foundation Ireland under Grant No 10/CE/B1821, Enterprise Ireland under Grant No CF/2011/1317, and Science Foundation Ireland (SFI) and Fraunhofer Gesellschaft under Grant No 16/SPP/3321.