Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip
Introduction
Fertilization in vitro (IVF) is a main method to treat infertility. The collected oocytes are fertilized with sperm in vitro; the fertilized eggs (embryos) are then cultured in a Petri dish before implantation (Reed, 2012). The embryo is cultured in a small droplet of culture medium. Then 2–3 selected mature embryos are implanted, based mainly on the morphologic features (Thurin et al., 2004; Schieve et al., 1999; López-Regalado et al., 2016; Martikainen et al., 2001). To identify further the embryo development, a non-invasive measurement of cytokines from the used culture medium is an attractive method. Several cytokines, so-called growth factors, act as signals of regulating functions during embryo-maternal interactions (Singh et al., 2011). Imbalances in expression levels and signaling of growth factors could lead to implantation failure or abnormal placentation (Guzeloglu-Kayisli et al., 2009). Some growth factors released in culture medium during culture have been suggested to be crucial for an embryo development, such as IL-1, IL-6, TNF-α etc. (Haynes et al., 1993; de Moraes‐Pinto et al., 1997; Robertson et al., 2000; Linjawi et al., 2004; Vujisic and Zidovec, 2005; Chung et al., 2000; Dadi et al., 2007; Lessey et al., 2002; Huang et al., 1997, 2005; Krüssel et al., 1998, 2006; Huang, 2006) TNF protein are widely present throughout pregnancy, where TNF-α is likely to have a role in growth and differentiation (Roby et al., 1990; Hunt et al., 1992; Argiles et al., 1997). Growth factor IL-1 is of particular interest during early embryonic development. IL-1β and IL-6 have been detected in an embryo culture medium; several studies indicated that levels of IL-1β correlate positively with the rate of implantation (Sheth et al., 1991; Baranao et al., 1997a, 1997b; Sequeira et al., 2015). In these studies, a conventional enzyme-linked immunosorbent assay (ELISA) was the main detection method, which requires a sample volume at least 50 μL, but the volume of the culture medium collected from a single embryo culture in current IVF is only 5–10 μL. To have sufficient sample volume, the used culture media from several embryos were hence typically aggregated for the measurement of growth factors; those measurement results became the average of several embryos, not from a single embryo culture medium. One study reported the results of measuring concentrations of IL-1β of different single-embryo culture media (Biba et al., 2015), but the IL-1β levels in many cases could not be detected. As dilution can provide a sufficient sample volume to meet the requirements of conventional ELISA, the demand on the limit of detection increases. A reliable detection of the growth factor from a single embryo culture medium of small concentration is thus still a challenge, apart from the detection of multiple growth factors from the rare culture medium.
Continuous-type microfluidic chips in biomedical applications typically consume a small sample volume with a short reaction duration (Karle et al., 2016; Lee et al., 2011). With digital microfluidics (DMF) (Lehmann et al., 2006; Sista et al., 2008), the required sample volume can be further decreased. For multiplex immunoassays (Mou and Jiang, 2017; Tighe et al., 2015; Vashist and Luong, 2019), the multiple biomarkers can be detected simultaneously from a sample solution to provide increased information with improved throughput. The multiplex immunoassays have extensively been demonstrated in continuous-type microfluidics (Ng et al., 2010), which however still need larger sample volume than digital microfluidic systems. There are some multiplex assays in EWOD systems by parallel processing (Lafrenière et al., 2014; Kalsi et al., 2017), i.e. measuring different targets from different droplets. Detecting multiple biomarkers from the same single droplet was not demonstrated in the EWOD device before.
Here we report a bead-based digital microfluidic chip developed by our group (Huang et al., 2015, 2016) for the simultaneous detection of two human embryo growth factors from the same single droplet. The detection principle is based on a fluorescent immunoassay. The bead-based digital microfluidic chip integrates droplet and bead manipulation techniques to achieve a small sample volume and a small limit of detection, allowing the detection of growth factors from a single embryo culture medium. The simultaneous detection can make more efficient use of a rare sample volume to obtain information from the culture medium, which can act as a powerful tool to study the growth factors of each embryo at various stages of culture. Human IL-1β and human TNF-α are demonstrated here to be simultaneously measurable from the same droplet of embryo culture medium. This medium is generally replaced with fresh medium on day 3; the embryo on day 5 or 6 is retrieved from the culture medium. In the current study samples of culture media retrieved on day 3 and day 5 or 6 serve to detect the growth factors, without affecting the standard clinical embryo culture. The chip manipulation techniques, and on-wafer protocols are described in detail as follows.
Section snippets
Fluorescent immunoassay for simultaneous detection
The simultaneous detection of two single-embryo growth factors (SEGF) is based on a fluorescent immunoassay. The concept of the on-chip immunoassay is shown in Fig. 1(a). The simultaneous detection of two growth factors (analytes) is based on the specific binding of the two analytes with distinct antibodies. The process begins with bead substrates of two types; each type contains magnetic beads conjugated with one specific capture antibody, as shown in Fig. 1(a1). The entire process includes
Single-analyte calibration curves
The calibration curve for each analyte is first derived using the proposed digital microfluidic chip with the corresponding bead substrates. The calibration curves of human IL-1β and human TNF-α, shown in Fig. 2(a) and Fig. 2(b), respectively, were prepared by measuring fluorescent intensities on standard solutions at six concentrations (0.0, 0.01, 0.1, 1, 10, 100 pg/mL). On-chip processes at each concentration were repeated three times. The limits of detection (LOD), the concentration
Conclusions
In IVF, some growth factors released in an embryo culture medium are suggested to be crucial for the embryo development, but the available volume from a single embryo culture medium is small, about 5–10 μL. The reliable detection of growth factors from a single embryo culture medium of small concentration hence remains a challenge. In our work not just one, but two growth factors from the same single droplet of embryo culture medium were detected for the first time with a bead-based digital
CRediT authorship contribution statement
Meng-Shiue Lee: Writing - original draft, Formal analysis. Wensyang Hsu: Writing - original draft, Formal analysis. Hong-Yuan Huang: Writing - original draft, Formal analysis. Hsueh-Yang Tseng: Writing - original draft. Chia-Tung Lee: Writing - original draft. Chung-Yu Hsu: Writing - original draft. Yi-Chueh Shieh: Writing - original draft, Formal analysis. Shih-Hung Wang: Writing - original draft. Da-Jeng Yao: Writing - original draft. Cheng-Hsien Liu: Writing - original draft.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by Ministry of Science and Technology, Taiwan, under Grant MOST 104-2211-E-009-124-MY2, MOST 105-2314-B-182A-114, MOST 106-3114-B-182A-002. Chang Gung Memorial Hospital funds, Taiwan, (CMRPG3G1361-2) also supported this research in part. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (CGMH-IRB numbers 104–9919A3 and 201700425B0).
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