Microfluidic Chromatography for Enhanced Amino Acid Detection at Ocean Worlds

Abstract

Increasing interest in the detection of biogenic signatures, such as amino acids, on icy moons and bodies within our solar system has led to the development of compact in situ instruments. Given the expected dilute biosignatures and high salinities of these extreme environments, purification of icy samples before analysis enables increased detection sensitivity. Herein, we outline a novel compact cation exchange method to desalinate proteinogenic amino acids in solution, independent of the type and concentration of salts in the sample. Using a modular microfluidic device, initial experiments explored operational limits of binding capacity with phenylalanine and three model cations, Na⁺, Mg²⁺, and Ca²⁺. Phenylalanine recovery (94-17%) with reduced conductivity (30-200 times) was seen at high salt-to-amino-acid ratios between 25:1 and 500:1. Later experiments tested competition between mixtures of 17 amino acids and other chemistries present in a terrestrial ocean sample. Recoveries ranged from 11% to 85% depending on side chain chemistry and cation competition, with concentration shown for select high affinity amino acids. This work outlines a nondestructive amino acid purification device capable of coupling to multiple downstream analytical techniques for improved characterization of icy samples at remote ocean worlds.

Keywords: Amino acid; Cation exchange chromatography; Desalination; Ocean world; Purification; Salt.

FIG. 1.

Microfluidic, cation exchange process for desalting a sample containing amino acids. Sequential syringes are introduced as follows: (1) 10 mM HCl to prepare and deprotonate the resin, (2) sample with amino acids that binds to the resin along with higher-affinity cations, (3) DI water to remove excess salts and contaminants, and (4) 1 M NH₄OH to selectively elute amino acids. A waste syringe collects eluent from steps 1–3, while the elute syringe collects step 4.

FIG. 2.

Decreasing phenylalanine recovery with increasing salt ratio. The vertical axis shows phenylalanine recovery as a percentage of amino acid introduced, and the horizontal axis shows the ratio between milliequivalents of cation introduced (Ca²⁺, Mg²⁺, and Na⁺) and phenylalanine introduced. Vertical bars represent eluted phenylalanine recovery range from a single experiment based on the 400 μL collection volume. The bottom marker indicates the full introduction of NH₄OH elution buffer to the chip, and the top marker indicates a subsequent DI water flush to remove remaining elution buffer from the chip.

FIG. 3.

Average recovery of 17 amino acids without salts in the 4 μM control experiment is independent of isoelectric point (pI) or amino acid side chain chemistry (R group). The horizontal axis plots amino acids by increasing pI, and colored bars identify amino acid R group. Values above the bars identify the mean value of triplicate runs, and vertical error bars represent one standard deviation in the positive and negative directions. Ala = alanine; Arg = arginine; Asp = aspartic acid; Cys = cysteine; Glu = glutamine; Gly = glycine; His = histidine; Ile = isoleucine; Leu = leucine; Lys = lysine; Met = methionine; Phe = phenylalanine; Pro = proline; Ser = serine; Thr = threonine; Tyr = tyrosine; Val = valine.

FIG. 4.

Average recovery of 17 amino acids in an ocean world analog sample is dependent on amino acid side chain chemistry (colored markers) rather than isoelectric point (horizontal axis order). With increased competition for binding sites, recovery percentages vary more widely between amino acids and sequential experiments. Vertical error bars represent one standard deviation in the positive and negative direction from the triplicate study. Abbreviations are the same as in Fig. 3.

FIG. 5.

Enrichment (mM recovered/mM introduced) of 17 amino acids in an ocean world analog sample is possible for several charged amino acids. The horizontal axis arranges amino acids by decreasing recovery percentage from Fig. 4 to demonstrate the impact of increased amino acid competition. Abbreviations are the same as in Fig. 3.