BioFluidix (Germany)

In this article, a quantitative liquid volume measurement method for the sub-nanoliter range using a quartz crystal microbalance (QCM) is described and experimentally analyzed. The primary measurement device to determine the volume of small liquid droplets is a QCM sensor coated with a surface-attached hydrogel to improve the mechanical coupling of the liquid to the sensor surface. An experimental evaluation of measured volumes in the range of 3 nl to 15 nl in normal room conditions has been performed with three identical sensors prepared with a PDMAA-1%MaBP hydrogel coating with a thickness of 1.5 µm ± 0.12 µm. A linearity of R2 more than 0.87, an average coefficient of variation (CV) within one experimental run of 5.7%, a mean absolute relative bias of 5.5%, and a sensor-to-sensor variation of 6.3% have been experimentally determined. The feasibility of this method has also been experimentally proven for the picoliter volume range down to 200 pl, with an average CV of 5.3% and a mean absolute relative bias of 6.5%. Furthermore, a stability evaluation consisting of 10 experimental series with approximately 150 measurements over the course of one week has been performed. This evaluation showed that the experimental setup, although exhibiting highly consistent performance within one measurement run, is not yet reproducible enough for long-term and repeated use because of undefined swelling and crack formation in the hydrogel layer. The low reproducibility implies a relatively high expanded uncertainty, with k = 2 according to the JCGM ‘Evaluation of Measurement Data—Guide to the Expression of Uncertainty in Measurement’ (GUM) for the total measurement method of approximately 3.82 nl when measuring a 10 nl liquid droplet. Nevertheless, the QCM method as described here contributes to significant progress beyond the state-of-the-art that might allow new opportunities for precise measurement of sub-nanoliter liquid volumes.

We present a novel measurement method based on the gravimetric principles adapted from the ASTM E542 and ISO 4787 standards for quantitative volume determination in the sub-microliter range. Such a method is particularly important for the calibration of non-contact micro dispensers as well as other microfluidic devices. The novel method is based on the linear regression analysis of continuously monitored gravimetric results and therefore is referred to as ‘gravimetric regression method (GRM)’. In this context, the regression analysis is necessary to compensate the mass loss due to evaporation that is significant for very small dispensing volumes. A full assessment of the measurement uncertainty of GRM is presented and results in a standard measurement uncertainty around 6 nl for dosage volumes in the range from 40 nl to 1 µl. The GRM has been experimentally benchmarked with a dual-dye ratiometric photometric method (Artel Inc., Westbrook, ME, USA), which can provide traceability of measurement to the International System of Units (SI) through reference standards maintained by NIST. Good precision (max. CV = 2.8%) and consistency (bias around 7 nl in the volume range from 40 to 400 nl) have been observed comparing the two methods. Based on the ASTM and ISO standards on the one hand and the benchmark with the photometric method on the other hand, two different approaches for establishing traceability for the GRM are discussed.

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.