The process of connecting genetic parts—DNA assembly—is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.

To alleviate ion suppression from phosphate buffer and to preserve separation integrity, a new capillary electrophoresis mass spectrometry (CE-MS) interface was developed. The interface consisted of a low-flow interface and a liquid junction. In this design, both the inlet reservoir and the liquid-junction reservoir were filled with phosphate running buffer. Because the phosphate anions in the column migrated toward the inlet reservoir (away from the electrospray ionization (ESI) source) the problem of ion suppression in ESI was avoided. The liquid junction was incorporated to eliminate issues of degraded separation observed when sheath liquid interfaces use different buffers for separation and MS analysis attributed to differences in anion velocity. The utility of the interface was demonstrated by the analysis of antihistamines at pH 3.5 and the analysis of perfluorocarboxylic acid at pH 9.5.

A double junction interface was utilized to preserve separation efficiency and alleviate ion suppression from sulfated β-cyclodextrin (S-β-CD) in electrokinetic chromatography-electrospray ionization-mass spectrometry. The utility of the approach was demonstrated by chiral EKC-MS analysis of dihydroxyphenylalanine and methyldihydroxyphenylalanine enantiomers using either low concentration (counter-migration mode; 0.1% S-β-CD) or high concentration (carrier mode; 2% S-β-CD). In the counter-migration mode, S-β-CD anions were supplied continuously from the junction reservoir to the separation column so that the effective separation length was preserved. This interface is especially useful under carrier mode in which high concentration of S-β-CD will migrate toward the ESI source. With the use of the double junction interface, the S-β-CD exited the separation column will remain in the junction reservoir, whereas the analyte will flow toward the ESI source through a connecting column. As a result, no ion suppression was observed and the sensitivity was improved significantly.

A preconcentration approach for CE-MS using counterflow-assisted electrokinetic injection was proposed. The proposed preconcentration method was based on a counterflow-compatible sheathless interface. The interface was fabricated using a capillary-assembled PDMS microdevice that allowed the application of a counterflow and provided liquid-film electrical conduction. During electrokinetic injection, a hydrodynamic counterflow was introduced into the separation capillary to retard the movement of the stacking boundary to the CE outlet. Accordingly, a long injection time was achieved without a loss of CE separation. With the use of reverse polarity mode and a dynamic polybrene-coated separation capillary (filled with a 1% formic acid solution), a counterflow-assisted electrokinetic injection of peptide samples (in a aqueous solution containing 1% ammonia and 50% methanol) for 10 min provided a sensitivity enhancement of 750-1480 and a detection limit of 20 pM for the five peptide standards. The proposed technique was applied to the analysis of low nanomolar concentrations myoglobin tryptic peptides.