RESEARCH ARTICLE
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Spectroscopic and Biophysical Methods to Determine
Diﬀerential Salt-Uptake by Primitive MembranelessPolyester Microdroplets
Chen Chen,* Ruiqin Yi, Motoko Igisu, Chie Sakaguchi, Rehana Afrin, Christian Potiszil,
Tak Kunihiro, Katsura Kobayashi, Eizo Nakamura, Yuichiro Ueno, André Antunes,
Anna Wang, Kuhan Chandru, Jihua Hao, and Tony Z. Jia*
𝜶-Hydroxy acids are prebiotic monomers that undergo dehydration synthesis
to form polyester gels, which assemble into membraneless microdroplets
upon aqueous rehydration. These microdroplets are proposed as protocells
that can segregate and compartmentalize primitive molecules/reactions.
Diﬀerent primitive aqueous environments with a variety of salts could havehosted chemistries that formed polyester microdroplets. These salts could be
essential cofactors of compartmentalized prebiotic reactions or even directly
aﬀect protocell structure. However, fully understanding polyester–saltinteractions remains elusive, partially due to technical challenges ofquantitative measurements in condensed phases. Here, spectroscopic andbiophysical methods are applied to analyze salt uptake by polyester
microdroplets. Inductively coupled plasma mass spectrometry is applied tomeasure the cation concentration within polyester microdroplets after
addition of chloride salts. Combined with methods to determine the eﬀects ofsalt uptake on droplet turbidity, size, surface potential and internal water
distribution, it was observed that polyester microdroplets can selectively
partition salt cations, leading to diﬀerential microdroplet coalescence due toionic screening eﬀects reducing electrostatic repulsion forces between
microdroplets. Through applying existing techniques to novel analyses related
to primitive compartment chemistry and biophysics, this study suggests thateven minor diﬀerences in analyte uptake can lead to signiﬁcant protocellular
structural change.

C. Chen, R. Yi, R. Afrin, Y. Ueno, T. Z. Jia
Earth-Life Science InstituteTokyo Institute of Technology
Meguro-ku, Tokyo 152-8550, Japan
E-mail: chenchen@elsi.jp; tzjia@elsi.jp

The ORCID identiﬁcation number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smtd.202300119© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction inany medium, provided the original work is properly cited.

1. Introduction
On prebiotic Earth, the heterogeneous
milieu contained a high diversity oforganic chemicals (and related reactions), including both biotic and abiotic compounds.[1,2] In particular, abiotic
molecules, i.e., those not commonly usedby extant biology, may have played an
important role in prebiotic chemistries
and could have assisted evolving primitive chemical systems.[2,3] 𝛼-Hydroxyacids (𝛼HAs), monomers with structures similar to 𝛼-amino acids, are one
type of abundant abiotic monomer likely
present on early Earth;[2,4] 𝛼HAs could
have arisen from plausible prebiotic
mechanisms such as terrestrial spark
discharge,[5] hydrothermal environment
reactions,[6] delivery via extraterrestrial
carbonaceous meteorites[7] or ultraviolet
photochemistry.[8]Solutions containing 𝛼HAs canundergo dehydration to yield gel-likepolyesters in conditions that mimic aqueous environments on early Earth.[9,10]
After rehydration in aqueous medium,
polyesters can assemble into membraneless droplets via liquid–liquid phase

M. Igisu, Y. Ueno
Institute for Extra-cutting-edge Science and Technology Avant-garde
Research (X-star)
Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
Yokosuka, Kanagawa 237-0061, Japan
C. Sakaguchi, C. Potiszil, T. Kunihiro, K. Kobayashi, E. Nakamura
The Pheasant Memorial Laboratory for Geochemistry andCosmochemistry
Institute for Planetary Materials
Okayama University
Misasa, Tottori 682-0193, JapanY. UenoDepartment of Earth and Planetary Sciences
Tokyo Institute of Technology
Meguro-ku, Tokyo 152-8551, Japan

DOI: 10.1002/smtd.202300119

Small Methods 2023, 7, 2300119

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separation (LLPS), a physicochemical phase transition phenomenon in which a homogeneous liquid spontaneously
demixes into two or more coexisting liquids.[11] Membraneless droplets, such as polyester microdroplets,[12,13] biomolecular coacervates[14–16] or aqueous two-phase systems (ATPS),[17]were ﬁrst proposed as model protocells by Oparin and Haldane in the 1920s[12,13,18–20] and in particular, membranelessprotocells are hypothesized to have preceded membrane-boundprotocells.[21]Membraneless protocells themselves exhibit a variety of dynamic behaviors that may indicate or be associated with life, suchas material exchange,[22] mobility,[23] coalescence and surface
wetting,[24] analyte concentration[25] and enhancement of compartmentalized analyte catalytic activity.[26] In fact, modern cells
also contain many membraneless organelles generated by LLPS
that have essential structural, catalytic and regulatory roles, whileaberrant intracellular phase separation can even cause neurodegenerative diseases.[27–29] Thus, the importance of membranelesscompartments in modern biology, coupled with the potential importance of membraneless protocells, suggests that LLPS may
structurally or functionally connect the origin of life to modern
biology.
Modern life is water-dependent, and given the existence of
diﬀerent aqueous environments on Earth, it has also been hypothesized that life originated and evolved in ancient aqueous
environments.[30] Thus, the structure of membraneless protocells would then depend sensitively on environmental factorsthat aﬀected primitive aqueous chemistries, such as temperature

A. Antunes
State Key Laboratory of Lunar and Planetary Sciences
Macau University of Science and Technology (MUST)
Taipa, Macau, SAR, China
A. Antunes, J. Hao, T. Z. Jia
Blue Marble Space Institute of Science
Seattle, WA 98104, USA
A. Wang
School of Chemistry
UNSW Sydney
Sydney, NSW 2052, Australia
A. Wang
Australian Centre for Astrobiology
UNSW Sydney
Sydney, NSW 2052, Australia
A. Wang
RNA Institute
UNSW Sydney
Sydney, NSW 2052, Australia
A. Wang
ARC Centre of Excellence for Synthetic Biology
UNSW Sydney
Sydney, NSW 2052, Australia
K. Chandru
Space Science Center (ANGKASA)
Institute of Climate Change
National University of Malaysia
Selangor 43650, Malaysia
J. HaoDeep Space Exploration Laboratory/CAS Laboratory of Crust-Mantle
Materials and Environments
University of Science and Technology of China
Hefei 230026, China

Small Methods 2023, 7, 2300119

and pressure,[31,32] salts[33] and pH,[34] etc. In particular, dissolved
salts are present in seawater, including those composed of cations
such as sodium (Na+ ), potassium (K+ ), magnesium (Mg2+ ), andcalcium (Ca2+ ) and anions such as chloride (Cl− ),[35] and would
have also been present in the ancient ocean,[36] although at different concentrations than the modern day. The same ions also
have multiple roles in modern life, including as electrical communication regulators (Na+ , K+ and Ca2+ ),[37] protein cofactors(Mg2+ )[38] or cellular stimulus-response controllers (Cl− ).[39]Salts not only aﬀect extant biology, but also aﬀect membraneless protocell properties. For example, the structure of biphasic
ssDNA–peptide–quaternized dextran coacervate droplets can betuned by NaCl, resulting in subcompartment fusion;[40] modulation of NaCl concentration drives the emergence of liquid crystal
phase transitions in dsDNA–peptide coacervates;[41] and membraneless protein droplets that generally phase separate only at
low KCl concentrations can also re-enter a phase separated state
at high KCl concentrations.[33] Previously, it was observed that
NaCl could induce polyester microdroplet coalescence, although
the mechanism is not yet known.[12] In general, how salt ions inﬂuence polyester microdroplet phase behavior or salt–polyesterinteractions in a prebiotic context and whether polyester microdroplets can selectively partition diﬀerent salts have not been wellstudied. Thus, we sought to characterize the ability for polyester
microdroplets to uptake salt ions, as well as the eﬀects of salt
uptake on physical microdroplet properties such as coalescenceand surface charge. Further elucidation of the physical properties of membraneless protocell models may help to answer the
many unanswered questions related to the use of membranelessphase-separated droplets as model protocell systems, such as the
emergence of “life-like” functions including partitioning, stability or evolution.[42]To mimic the messy prebiotic chemical milieu, we ﬁrst
subject a series of 𝛼HAs to dehydration synthesis to form
polyesters, which were then subsequently screened for microdroplet assembly.[43] Based on their signiﬁcant propensity toform droplets after synthesis, dl-3-phenyllactic acid (PA) waschosen as the model 𝛼HA of choice for this study.[12] However, given that salt–polyester interactions (which could govern
salt uptake) may be signiﬁcantly aﬀected by electrostatic interactions, incorporation of basic or acidic 𝛼HA residues into apolyester chain could result in diﬀerential interactions with salts,
as compared to a fully neutral polyester such as poly(PA). Previously, primitive polyesters containing basic (S)-(-)-4-amino-2hydroxybutyric acid (4a2h) and acidic dl-malic acid (Malic) has
been demonstrated.[13,44] Thus, our 𝛼HA library consisted of PA,
4a2h and Malic, and we produced polyester microdroplets composed of both homopolyesters (poly(PA)) and potentially charged
heteropolyesters (poly(Malic-PA) and poly(4a2h-PA)) for downstream analysis.
Inductively coupled plasma mass spectrometry (ICP-MS) can
detect ions or metals (e.g., in extraterrestrial asteroid samples)and can simultaneously measure multiple elements with highsensitivity.[45] We thus applied a novel ICP-MS-based analytical
approach to determine the cation distribution within polyester
microdroplets following incubation with four diﬀerent chloride
salts (NaCl, KCl, MgCl2 and CaCl2 ; all present in early oceans).
We also conducted in parallel a series of biophysical measurements on the turbidity, size, water distribution and surface

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Figure 1. a) Chemical structure of the three 𝛼HAs studied. b) Polyester synthesis from dehydration of 𝛼HA monomers. c) Polyester microdroplet
formation after rehydration and their coexistence with and/or uptake of salt ions.

potential of the droplets to correlate the physical eﬀects resulting
from salt uptake within the polyester microdroplets (Figure 1).
We observed that polyester microdroplets can selectively partition
salt cations, leading to diﬀerential microdroplet coalescence due
to ionic screening eﬀects reducing electrostatic repulsion forces
between microdroplets. To our knowledge, this is the ﬁrst application of ICP-MS to understanding the biophysics of a nonassociative membraneless protocell system (i.e., polyester microdroplets) in the origins of life ﬁeld. These ﬁndings suggest thateven minor diﬀerences in analyte uptake by primitive compartments could result in signiﬁcant protocellular structural changes,
leading to potential chemical evolution of protocell systems inprebiotic aqueous environments.

2. Results and Discussion
2.1. Polyester Dehydration Synthesis and Microdroplet Assembly
via Rehydration
Previous studies have shown that synthesized polyesters composed of poly(PA) and 1:1 4a2h/PA can undergo droplet assembly. Dehydration synthesis of pure 4a2h resulted in polymerization, but not microdroplet formation,[13] while increasing amounts 4a2h incorporated into PA-containing polyesters
resulted in increased solubility and a lower tendency of droplet
assembly.[13] However, polyester droplets composed of Malic/PA
have not yet been demonstrated. We thus subjected pure PA
and Malic solutions, as well as 1:1 Malic/PA and 1:1 4a2h/PA

Small Methods 2023, 7, 2300119

solutions, to dehydration synthesis to generate homo- and heteropolyester products. The resulting polyester products reachedlengths of up to 20 residues depending on the droplet composition as analyzed by matrix-assisted laser desorption ionization time-of-ﬂight mass spectrometry (MALDI-TOF-MS). The
homopolyesters poly(PA) and poly(Malic) were detected frompure PA or Malic monomer reactions (Figure 2a; Figure S1 andTables S1 and S2, Supporting Information). The heteropolyesters
poly(Malic-PA) and poly(4a2h-PA) were also detected as productsof the respective reactions starting from mixed monomer solutions (Figure 2b,c; Tables S3 and S4, Supporting Information),suggesting co-incorporation of all monomers present in the initial reaction mixtures.
We then characterized the phase separation properties ofthe synthesized polyesters upon rehydration in aqueous solution. Similar to what was previously reported for poly(4a2h),poly(Malic) did not form droplets upon aqueous rehydration,
likely due to the high solubility of the poly(Malic) polymers.
Rehydration of poly(PA), poly(Malic-PA) and poly(4a2h-PA) resulted in turbid solutions (Figure S2, Supporting Information)
composed of spherical microdroplets of size from a few micrometers up to tens of micrometers in diameter (Figure 2).
Thus, we showed successful droplet assembly with three different polyester systems: a neutral polyester poly(PA), an anionic residue-containing polyester poly(Malic-PA) and a cationic
residue-containing polyester poly(4a2h-PA). These three systems are suﬃciently diﬀerent in charge and will be used toprobe potential salt–polyester interactions, which may depend

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Figure 2. Representative MALDI-TOF-MS spectra showing synthesis and 𝛼HA incorporation for a) poly(PA), b) poly(Malic-PA) and c) poly(4a2h-PA).
Zoom-in region insets for (b) and (c) are included for ease of visualization of the product compositions and Malic and 4a2h incorporation, respectively.
Peak lists can be found in Tables S2–S4 (Supporting Information). Images showing spherical microdroplets formed from poly(PA), poly(Malic-PA), andpoly(4a2h-PA) condensed phases in aqueous media visualized by optical microscopy are also included. Scale bars are 100 μm.

on electrostatic interactions. We also note that the observation ofpoly(Malic-PA) microdroplets is the ﬁrst demonstration of primitive polymerization and assembly of acidic residue-containing
polyester microdroplets. Similar to the 4a2h/PA system, we expect that increasing the Malic/PA monomer ratio prior to dehydration synthesis would also result in increased solubility and asubsequent decrease in droplet assembly propensity.
Up to 100 × 10−3 m NaCl, KCl and MgCl2 , and up to 10 ×−3
10 m CaCl2 did not appear to inhibit polyester polymerization
or microdroplet assembly (Figures S3 and S4, Supporting Information). While even further increases in salt concentration (inthe hundreds of millimolar range for NaCl in the early Ocean,

Small Methods 2023, 7, 2300119

for example[46] ) or presence of other salts could aﬀect polyester
polymerization or droplet assembly, it is out of the scope of thisstudy to probe these boundaries. Thus, subsequent experimentsin this study probed individual salt concentrations only up to
100 × 10−3 m.
We next set out to investigate changes in structure or coalescence of poly(PA), poly(Malic-PA) and poly(4a2h-PA) droplets
induced by the four chloride salts. None of the three droplet systems completely dissolved or disassembled upon incubation with
100 × 10−3 m of each salt (Figure S5, Supporting Information).
This suggests that the droplets are kinetically trapped, rather
than equilibrium structures; addition of 100 × 10−3 m CaCl2 to2300119 (4 of 13)© 2023 The Authors. Small Methods published by Wiley-VCH GmbH

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preformed droplets did not result in disassembly, whereas
dehydration/rehydration of 𝛼HAs with 100 × 10−3 m CaCl2 did
not lead to droplet formation. However, the number of droplets
qualitatively appears to have decreased upon salt addition.
This observation matches previous observations of salt-induced
polyester droplet coalescence, which would theoretically decrease
the number of droplets, but simultaneously increase the average
droplet size.[12] These changes in droplet properties were likely
due to the salts themselves, rather than by the dilution process incurred when salt solutions are added to the polyesters, as dilution
in pure water does not qualitatively appear to change the numberof droplets present (Figure S6, Supporting Information).

2.2. Salt-Induced Changes in Polyester Microdroplet Number,Size, and Surface Potential
Since the glass substrates used for microscopy can aﬀect droplet
coalescence and introduce artifacts for quantiﬁcation of droplet
properties, we sought to use turbidimetry for quantitative droplet
analysis. Assuming that a decrease in turbidity from droplets
spontaneously decreasing in size is not possible, we use opticaldensity at 600 nm as a proxy for droplet density within a solution. We ﬁrst tracked the turbidity of the assembled droplets over
two hours (to minimize the possibility of polyester hydrolysis anddroplet disassembly at very long timescales[12] ) in the presence orabsence of salts.
In the absence of salts, the turbidity of poly(PA) microdroplets decreases slightly over two hours, whereas the turbidityof poly(Malic-PA) and poly(4a2h-PA) microdroplets decreases ina more pronounced manner over the same two hours (Figure S7,
Supporting Information). This suggests that while there is somedecrease in droplet density over time (perhaps due to coalescence or sedimentation), droplets are still relatively stable over
two hours. However, poly(Malic-PA) and poly(4a2h-PA) droplets
appear to decrease in number more rapidly compared to poly(PA)droplets. This could be due either to increased coalescence, or increased solubility of poly(Malic-PA) and poly(4a2h-PA) droplets.
Indeed, incorporation of hydrophilic Malic or 4a2h into neutral,
apolar PA-containing polyesters could lead to greater droplet solubility via weakening of noncovalent interactions, such as the hydrophobic eﬀect, the likely main driving forces for microdroplet
assembly.[12,13] Alternatively, polyesters with some hydrophilic
𝛼HAs could modulate the surface charge of the droplets, resulting in changes to colloidal stability (which will be discussed laterin this study).
We observed that for poly(PA), addition of up to 10 × 10−3 m
NaCl or KCl or up to 1 × 10−3 m MgCl2 or CaCl2 did not affect droplet density. However, incubation of poly(PA) with 100 ×10−3 m NaCl or KCl or with ≥10 × 10−3 m MgCl2 or CaCl2 , resulted in a rapid decrease in turbidity (Figure 3a). We observed
similar trends upon incubation of poly(Malic-PA) and poly(4a2hPA) with the salts. However, poly(Malic-PA) showed some sensitivity to the monovalent salts as low as 10 × 10−3 m and to thedivalent salts as low as 1 × 10−3 m, while poly(4a2h-PA) is sensitive to 10 × 10−3 m NaCl, but not KCl (Figure S8, Supporting
Information). These observations suggest that higher concentrations of and higher valence salts result in greater decreases indroplet number over time.

Small Methods 2023, 7, 2300119

We thus sought to conﬁrm whether the decrease in droplet
number upon salt addition was due to coalescence by performing time-dependent dynamic light scattering (DLS) assays, enabling direct measurement of particle size as a proxy of droplet
coalescence. We observed that in the absence of salts, droplet
size increased only slightly, suggesting that little coalescence occurs in the absence of salts (Figure S9, Supporting Information).
Upon addition of the salts, higher concentrations of salts generally resulted in greater increases in droplet size (indicating coalescence) over time. For example, 100 × 10−3 m NaCl generally resulted in more rapid coalescence than 10 × 10−3 m NaCl
(Figure 3b; Figure S10, Supporting Information). Similarly, divalent salts typically required lower concentrations to eﬀect droplet
coalescence as compared to monovalent salts. For example, 100 ×10−3 m NaCl was required to drive poly(PA) coalescence, while
only 10 × 10−3 m MgCl2 resulted in a similar result (Figure 3b).
We note that this observation does not strictly follow the SchulzeHardy rule in DLVO theory,[47] which simplistically suggests thata divalent salt with a 64-fold lower concentration than that ofa monovalent salt would induce the same result for a colloidal
system. Speciﬁcally, although 100 × 10−3 m NaCl resulted inpoly(PA) coalescence, 10 × 10−3 m MgCl2 was required to eﬀect
the same result. However, the Schulze-Hardy rule suggests that
only 1.56 × 10−3 m MgCl2 would have been required. We also
note exceptions in poly(Malic-PA), where 10 × 10−3 m NaCl could
drive coalescence, while CaCl2 required concentrations greaterthan 10 × 10−3 m to drive coalescence (Figure S10, Supporting
Information). These discrepancies between experimental results
and theoretical predictions point to the fact that polyester droplets
are likely not colloids.
We next compared the trends in the changes in polyester
droplet number and size upon salt addition, and deduced that decreases in droplet number (Figure 3a; Figure S8, Supporting Information) were likely due to salt-induced coalescence (Figure 3b;
Figure S10, Supporting Information). While it is possible thatdroplet swelling due to salt uptake led to an increase in droplet
size, the fact that a concurrent decrease in droplet number occurred leads us to believe that the main driver of droplet growthis coalescence. We thus hypothesized that the mechanism of saltinduced droplet coalescence may be due to modulation of droplet
surface charge by uptaken salts, which could result in greater
ease of interfacial interactions leading to coalescence. Thus, wesought to measure salt-induced changes in the surface potentialof microdroplets. We observed through zeta-potential measurements that the surface charge in the absence of salts for all three
droplet systems was slightly negative, with the poly(PA) droplet
surface retaining more negative charge than poly(4a2h-PA) andpoly(Malic-PA) droplet surfaces (Figure 3c; Figure S11, Supporting Information). Negatively charged droplet surfaces would leadto repulsion between droplets rather than coalescence. The negative surface charge is potentially due to accumulation of hydroxide ions at the droplet surface. Low pH systems (our system isaround pH 3) lead to existence of free hydroxide ions, which appear to have some aﬃnity to droplet or phase-separated boundaries of apolar assemblies.[48] There may also be some contribution from potential negative charges on the terminal carboxylgroups of product polyesters, which can be negatively charged
depending on the ambient pH. For example, the pKa of the PA
carboxyl group is 3.46,[49] and a pH of ≈3 results in ≈25% of the2300119 (5 of 13)© 2023 The Authors. Small Methods published by Wiley-VCH GmbH

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Figure 3. a) Normalized turbidity (OD600) and b) normalized particle size (DLS) was measured over 2 h for poly(PA) microdroplets in the presenceof chloride salt solutions (NaCl, KCl, MgCl2 , and CaCl2 ranging from 1, 10, to 100 × 10−3 m). Both turbidity and particle size at each time point werenormalized to the initial average value before incubation (time = 0), respectively, for poly(PA) microdroplets mixed with the appropriate volume of water.
c) Average and standard deviation of the surface potential of poly(PA) microdroplets in the presence of water or 100 × 10−3 m of the four chloride salt
solutions. d) Relationship between the average and standard deviation of the surface potential of poly(PA) microdroplets upon incubation with the fourchloride salt solutions at concentrations up to 100 × 10−3 m. e) Schematic diagram for salt-driven polyester microdroplet coalescence. n = 3 for OD600
and DLS experiments; n = 6 for surface potential experiment reported herein. Error bars indicate standard deviation.

terminal carboxyl of the product polyesters being deprotonatedand retaining a negative charge. Alternatively, previous studies
suggest ≈15%–20% unreacted 𝛼HA monomer remains after thesynthesis reaction, some of which could also retain a negative
charge.[20] Some unreacted, deprotonated monomers could also
be present at the droplet surfaces.
We attribute the slightly weaker negative surface charge ofpoly(4a2h-PA) and poly(Malic-PA) droplets to the fact that theirinteriors may be less apolar than poly(PA) droplets, due to incorporation of basic or acidic residues. This could result in a greater
propensity for free hydroxide anions to reside within the less apolar droplet interior, as opposed to the droplet surface, thus decreasing accumulation of negative surface charge.[48] Poly(4a2h-

Small Methods 2023, 7, 2300119

PA) droplets may also have protonated amino groups within incorporated 4a2h residues, contributing to even more neutralization of its surface charge compared to poly(PA).
We then observed that addition of 100 × 10−3 m of each of
the four chloride salts resulted in signiﬁcant neutralization of
the surface charge of all polyester droplet systems, in some cases
reaching nearly 0 mV (Figure 3c; Figure S11, Supporting Information). The droplet surface charge neutralization also scaledwith increasing salt concentrations (Figure 3d; Figure S12, Supporting Information). We surmise that the salt-induced neutralization of droplet surface charge led to less electrostatic repulsion of the originally negatively charged droplet surfaces, which
is what drives polyester microdroplet coalescence (Figure 3e).

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Figure 4. a) ICP-MS measurement of salt cation concentration (Na+ , K+ , Mg2+ , and Ca2+ ) following incubation of poly(PA) droplets with diﬀerent
concentrations of the salt solutions indicated inside (“Droplet”) and outside (“Supernatant”) of the droplet. “Initial Mixture” indicates the sample
before separation of the droplet and supernatant. In the sample with 1 × 10−3 m NaCl added, the initial Na+ concentration was greater than 1 ×
10−3 m due to detected Na+ contaminants in the PA starting materials (Table S13, Supporting Information). *p < 0.05; **p < 0.01 (see Table S14 in the
Supporting Information for relevant p-values). b) Partition coeﬃcient of poly(PA) homopolyester microdroplets for all salt cations at concentrations up
to 100 × 10−3 m. c) Partition coeﬃcient of poly(PA), poly(Malic-PA) and poly(4a2h-PA) microdroplets for all salt cations at 100 × 10−3 m. n = 3 for all
experiments reported herein. Error bars indicate standard deviation.

2.3. Salt Ion Uptake by Polyester Microdroplets
We next sought to quantitatively measure the amount of eachsalt uptaken by the polyester microdroplets by applying ICP-MS
analysis to the droplets. ICP-MS is commonly used for elementspeciﬁc detection and quantiﬁcation, especially in geochemistry
and planetary science,[50,51] and can measure salt ion concentration within the droplets following uptake. ICP-MS analysis wasable to detect each of the four cations of the chloride salts (Na+ ,K+ , Mg2+ and Ca2+ ), but could not detect chloride anions due
to poor ion formation eﬃciency of Cl− in the argon plasma andisobaric interference.[52] Thus, we focused only on quantiﬁcationof cation uptake. We ﬁrst measured the ambient cation content
in the 𝛼HA monomer solutions and polyester microdroplets inthe absence of salts, and found that Na+ was generally present at
higher initial amounts compared to the other three ions in all systems other than pure 4a2h (Table S13, Supporting Information).
We suspect this to be due to minor NaCl or sodiated monomer
counterion impurities in the original commercially available PA
and Malic monomer samples. However, we reckoned that thesmall scale of these impurities (Table S13, Supporting Information) would not signiﬁcantly aﬀect any of the physical eﬀects observed and thus proceeded without removal of Na+ impurities.

Small Methods 2023, 7, 2300119

We then mixed fresh poly(PA) homopolyester microdropletswith each of the four salts at concentrations up to 100 × 10−3 m,followed by centrifugation to separate the droplets from the surrounding solution (“supernatant”). After ICP-MS analysis, wefound that in all cases, the concentrations of the cations in the
supernatants are nearly identical to that measured in the initial
mixtures before droplet/supernatant separation (Figure 4a). Thisis likely due to the fact that the volume occupied by the dropletsis small compared to the supernatant. Even if there is a large
amount of salt uptaken by the droplets, the overall concentrationof salt cations in the supernatant will not change signiﬁcantly. We
also detected each cation within the poly(PA) droplets, suggesting that the poly(PA) droplets can uptake each salt cation to somedegree (Figure 4a). For all cases, the salt cation concentration in
the droplets was signiﬁcantly diﬀerent compared to in the supernatant (Figure 4a). This suggests that the salt uptake process islikely selective and diﬀerential rather than nonselective, as would
have been the case if the salt cation concentration inside and outside of the droplets was equivalent. As the droplets were incubated with salt overnight, we also believe that the measurements
here are likely at equilibrium, and that the data indicate selective
salt uptake rather than reﬂecting incomplete salt uptake due todiﬀerences in salt uptake rates.

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Upon closer examination, K+ was generally uptaken by the
droplets the most, with Mg2+ uptaken the least. Furthermore,
the relative amount of cations in solution uptaken was greater atlower salt concentrations (Figure 4a). At 1 × 10−3 m, we observedthat K+ could in fact be concentrated within poly(PA) droplets. To
visualize the ability of the droplets to uptake salts diﬀerentiallymore clearly, we deﬁne a term called partition coeﬃcient, whichis the ratio of the cation concentration inside of the droplets
versus the supernatant. A higher partition coeﬃcient suggests
greater salt uptake. A partition coeﬃcient >1 also reﬂects the ability of the droplet to concentrate the uptaken salt from solution.
We observe that for all cations, increasing solution concentration resulted in a decrease of the partition coeﬃcient in poly(PA)(Figure 4b). This suggested that the droplets may have some upper limit for salt cation uptake, whether due to some physical orchemical limitation. Even increasing the total amount of salt insolution may not allow the droplets to uptake more cations than
this upper limit. However, this upper limit may be diﬀerent foreach cation, and we may not have reached the limit in this study.
Poly(PA) microdroplets appear to diﬀerentially uptake salt
cations in the following order of decreasing aﬃnity: (K+ > Ca2+
> Na+ > Mg2+ ) (Figure 4b). The exact mechanism of this diﬀerence is not clear, and may be due to a number of factors suchas diﬀerences in cation structure, size[53] and/or binding aﬃnity
with poly(PA). However, the order of decreasing aﬃnity of cation
uptake matches roughly with the order of decreasing ionic radius (K+ > Ca2+ = Na+ > Mg2+ ). If we consider just the divalentcations, Mg2+ has a larger hydration sphere than Ca2+ .[54] A larger
hydration sphere could weaken the electrostatic interaction of thecation interior with the negatively charged polyester microdroplet
surface, resulting in lower Mg2+ uptake compared to Ca2+ . For
the monovalent cations, Na+ has a larger hydration sphere thanK+ , which could result in greater K+ uptake compared to Na+ .
There may also be prebiotic evolutionary advantages forpolyester droplets to selectively incorporate of K+ over other salts,
such as for generation or maintenance of protocell boundary
chemical potential. We make this conjecture considering that intracellular K+ concentration is much higher than that of Na+ ,Mg2+ and Ca2+ in plant and animal cells, likely due to the actionof potassium channels or the potassium-sodium pump, respectively. For instance, plants maintain a high (100–200 × 10−3 m) K+
and a low (1–10 × 10−3 m) Na+ concentration in their cytosol.[55]Animals also maintain a high ≈150 × 10−3 m K+ and a low ≈15 ×
10−3 m Na+ concentration.[56] However, the ancient ocean likely
contained much more Na+ than K+ ,[57] with some estimates suggesting up to 1100 × 10−3 m Na+ .[58] Polyester microdroplets inancient oceans could selectively uptake low-abundance K+ , while
simultaneously excluding high-abundance Na+ . This may havebeen one mechanism to achieve a local protocellular microenvironment containing a K+ /Na+ ratio more on par with that observed in modern biology.
Salt–polyester interactions could be one driver for salt uptake,and polyester droplets with acidic or basic residues could leadto diﬀerences in salt uptake. We thus subjected poly(Malic-PA)
and poly(4a2h-PA) microdroplets incubated with 100 × 10−3 msalt solutions to ICP-MS analysis to measure salt cation uptake.
In nearly all cases, poly(Malic-PA) and poly(4a2h-PA) droplets
appeared to uptake more of all cations than poly(PA) droplets(Figure S13 and Table S14, Supporting Information). We at-

Small Methods 2023, 7, 2300119

tribute these observations to the fact that incorporation of 4a2h
and Malic into an otherwise neutral polymer may increase the internal polarity of the droplets, allowing ions to have more aﬃnity to the droplets. Based on partition coeﬃcient analysis, the order of salt cation uptake aﬃnity was also similar for all systems(Figure 4c and Table S14, Supporting Information).
Surface charge measurements (Figure 3c,d; Figures S11 andS12, Supporting Information) suggested that incubation with
100 × 10−3 m of each salt nearly neutralized the surface chargeof all droplets. However, the ICP-MS studies suggest that onlya small portion of the cations in solution are uptaken by the
droplets in some cases. We speculate that the cations may preferentially localize to the droplet surface, rather than being evenly
distributed throughout the entire droplet. Thus, only a small concentration of salt needs to be uptaken by the droplet to completely neutralize the surface charge. This could be due to thehydrophilic character of the uptaken cations, having aﬃnity for
aqueous solution over the interior of the apolar droplets.[12,13]
However, the interior of the droplets cannot be fully anhydrous,and so some salts should still reside within the droplet interior,albeit possibly at lower concentrations than at the surface. Indeed, microRaman analysis was able to detect the presence ofwater and ester bonds within the polyester droplets (Figures S14
and S15, Supporting Information). However, the apparent relative amount of water within the droplets appeared to be less thanthat the surrounding solution, reﬂecting the apolar nature of thedroplet interior (Figures S14 and S15, Supporting Information).
The addition of salts also did not signiﬁcantly aﬀect the water
signal intensity within the droplets (Figures S14 and S15, Supporting Information). This suggests that while there is still somewater within the droplets, the fairly apolar character of the droplet
interior is still dominant even in the presence of salts.

3. Conclusion
In this work, we applied existing spectroscopic and biophysicalmethods to perform novel physical characterization of the saltmediated dynamic phase behavior of polyester microdroplets. We
found that polyester microdroplets can be assembled from systems that incorporate 𝛼HAs with acidic functional groups (i.e.,
Malic) and from reactions that contain chloride salts (up to 100 ×10−3 m in some cases). These results further increase the breadthof the primitive chemical repertoire capable of forming polyester
microdroplets. This is relevant as the early Earth may have contained a large diversity of potential ester-forming monomers, including not only 𝛼HAs, but also heterocyclic monomers which
can form polyesters through ring-opening polymerization.[59]The early Earth environment may also have been diverse in salinity, ranging from pure freshwater to oceanic environments to underwater hypersaline underocean brines that could have been thecradle of eventual halophile emergence.[60,61]
Additionally, we discovered that salts could drive polyester microdroplet coalesce. We propose that the high salt concentrations
encountered in the primitive marine environments mentioned
above may drive polyester microdroplet coalescence through neutralization of the typically negatively charged droplet surface. Thisin turn weakens the electrostatic repulsive forces at the droplet
surface due to potential ion localization at the surface and inhibits
droplet-droplet repulsion, driving coalescence. Such salt-induced

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droplet coalescence could have been one driver of primitive protocellular recombination, one mechanism by which compartmentalized replicating informational polymers could have encountered greater genetic diversity. Such a phenomenon could havebeen one mechanism of primitive horizontal gene transfer, while
also promoting polyester compositional recombination as well.
It was also previously observed that vortexing and sonication can
result in division of droplets in the laboratory.[12] Cycles of saltinduced droplet coalescence interspersed with environmentally
driven cell division could plausibly be achieved through turbulent waves caused by storms or fumarole-induced mixing in hot
springs on early Earth. Such an environment could be conducive
to chemical evolution of polyester microdroplets, whether via genetic evolution of encapsulated informational polymers or via
compositional evolution of the polyesters themselves.[62]Furthermore, ICP-MS analysis revealed that polyester microdroplets uptake salt cations in a diﬀerential manner. ICP-MS isoften used for materials analysis, such as in geochemistry andplanetary science, and has previously been used for analysis ofassociative coacervates.[63] Here, we extended this notion and analyzed dissociative membraneless polyester protocell models. We
observed that uptake of only a small amount of cations could result in complete neutralization of the droplet surface charges,
suggesting that uptaken salts may preferentially reside at the
droplet interface. However, it is unclear whether salt uptake isdriven by adsorption or absorption. Further studies relating salt
uptake with droplet volume are suggested to rule out one or
the other possibility. While the exact mechanism of diﬀerential
salt uptake is still unclear, we speculate that it is related to theionic radius and hydration shell size of the cations. Nevertheless, we observed that changes in the primary composition of
the polyesters could be one method to modulate cation uptake
into polyester droplets. This result is also related to our previous observation that polyester primary composition changes canresult in acquisition of an RNA segregation ability by polyester
droplets.[13] Further investigation of the mechanism and evolutionary advantages of the observed diﬀerential salt uptake will beneeded to holistically understand protocell-encapsulated primitive reactions that require free ions as cofactors. For example,
Mg2+ is essential for RNA folding, ribozyme catalysis and nonenzymatic RNA replication,[64–66] and understanding the feasibilityof such processes within the polyester droplets is necessary. Similarly, whether encapsulated Mg2+ (or another cation) could modulate nucleic acid uptake into the droplets due to salt-nucleic acidbinding, or vice versa, is also worth a detailed follow-up study inthe future.
Finally, the study of the eﬀects of diﬀerent types of salt on
membraneless polyester microdroplets could also be relevant to
the origin and existence of potential life oﬀ of Earth. For example,
aqueous environments and high salinity are relatively common
in inner Solar planetary bodies (e.g., Mars)[67–70] or even the exooceans of the icy moons of the outer solar system such as Europa or Enceladus.[71,72] 𝛼HA monomers could also have been
existed on Mars or the icy moons via meteoritic delivery,[7,73–74]
especially on Mars, where extramartian organic delivery via meteorites has been observed.[75] Experimental demonstration of reducing chemistries simulating those of Mars or Enceladus conditions has also demonstrated the potential synthesis of 𝛼HAmonomers in those environments.[76,77] Finally, evaporation of

Small Methods 2023, 7, 2300119

surface liquid water in the past on Mars,[78] dehydration synthesis
in ice on Europa[79] or low-water activity pores on Enceladus[80–82]
could provide appropriate conditions for the dehydration synthesis chemistries presented herein. Nevertheless, we do acknowledge that 𝛼HAs have not been directly detected on Mars or theicy oceans of extraterrestrial bodies, perhaps owing to instrument
detection limits or preservation issues,[83–85] and thus it cannot be
ruled out that 𝛼HAs exist on other extraterrestrial bodies. Thus,
further development of more sensitive or novel methods to analyze and characterize 𝛼HAs, polyesters, and their subsequent
microdroplets in extraterrestrial environments will be necessary
to more clearly reveal the relevance of polyester-based protocell
systems to the origins of life both on and oﬀ Earth.

4. Experimental Section
Chemicals: PA (dl-3-phenyllactic acid) was purchased from SigmaAldrich (St. Louis, MO, USA). 4a2h ((S)-(-)-4-amino-2-hydroxybutyric acid)and Malic (dl-malic acid) were purchased from Tokyo Chemical Industry
Co. (Chuo-ku, Tokyo, Japan). All chloride salts (NaCl, KCl, MgCl2 ⋅6H2 O
and CaCl2 ⋅2H2 O) were purchased from Nacalai Tesque (Nakagyo-ku, Kyoto, Japan). All other chemicals, including acetonitrile, were purchasedfrom Sigma-Aldrich.
Dehydration Synthesis of Polyesters: All syntheses were carried out in
13 mm borosilicate test tubes (Thermo Fisher Scientiﬁc, Minato-ku, Tokyo,
Japan) or 1.5 mL microcentrifuge tubes (Eppendorf, Chiyoda-ku, Tokyo,
Japan), starting from a total volume of 500 μL of monomer solution anda total starting monomer concentration of 500 × 10−3 m. This meant that
all homopolyesters started with 500 μL of a 500 × 10−3 m stock solution,
whereas heteropolyester cases (1:1 Malic/PA and 1:1 4a2h/PA) started
with 250 μL each of both stock solutions at 500 × 10−3 m, i.e., 250 μL
of a 500 × 10−3 m PA stock solution was mixed with a 500 × 10−3 m Malic
stock solution (resulting in 250 × 10−3 m of each monomer after mixing).
In addition, for polymerization in the presence of salts, 250 μL of a 500 ×10−3 m PA stock solution was mixed with 250 μL of NaCl, KCl, MgCl2 or
CaCl2 with ﬁnal salt concentrations of 10 × 10−3 or 100 × 10−3 m after
mixing. All stock solutions were prepared in ultrapure water (18.2 MΩ cm
and <5 ppb total organic carbon, Barnstead Smart2Pure system, Thermo
Fisher Scientiﬁc) after vortexing and sonication without adjustment of pH.
Stock solutions were stored at 4 °C until use. The PA stock solution did not
fully solubilize without repeated heating/vortexing cycles; when preparing
PA stock solution, the solution was heated to 60 °C on a heating plate withoccasional vortexing to fully solubilize PA in solution, followed by coolingto room temperature.
The synthesis reactions were allowed to dry uncovered for 1 week atconstant 80 °C using Sahara 310 or 320 dry heating baths (Rocker Scientiﬁc, New Taipei City, Republic of China) in a draft chamber to mimica primitive dehydrating environment. After 1 week, the samples werecompletely dry and formed a gel-like state. All macroscopic condensed
phase photographs were collected with a Huawei Mate 10 Pro (Shenzhen,
Guangdong, China).
Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS): MALDI-TOF-MS was performed using an ultraﬂeXtreme Bruker Daltonics (Billerica, MA, USA) MALDI-TOF-MS in positive ion mode. External mass calibration was conducted using standard peptide mixtures provided by the user facility. Poly(Malic) was
dissolved in water, whereas poly(PA), poly(Malic-PA) and poly(4a2hPA) were dissolved in tetrahydrofuran (THF) (10 mg mL−1 ). CHCA
(alpha-cyano-4-hydroxy-cinnamic acid) was chosen as the sample matrix for all samples, except poly(PA) polymerized with 10 and 100 ×10−3 m CaCl2 used DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2propenylidene] malononitrile) as the matrix after optimization due to poor
spectral quality when CHCA was used as the matrix.
The sample matrix was ﬁrst dissolved in THF. Dissolved samples werethen mixed with the matrices (1:10 (v/v)) and applied to the plate before

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analysis. Monoisotopic peaks were then identiﬁed by isolating the highest
intensity peak in an isotope envelope corresponding to a polymer productusing a peak list generated from mMass (Open Source Software, Prague,
Czech Republic) to subtract the matrix background signal. Major peaks
were isolated by applying the following parameters: S/N > 10, absolutepeak intensity >1000 and baselining (100 precision and 0 relative oﬀset).
Mass accuracy in ppm was calculated by comparing the observed mass
with the calculated mass for major peaks corresponding to polymerization products. Detailed peak lists are presented in Supporting Information; generally, only sodiated peaks (which are the most abundant and effectively demonstrate stepwise residue addition on a growing polyester
polymer chain) are reported unless otherwise noted.
Droplet Formation: All synthesized polyesters were ﬁrst rehydrated in
500 μL 4:1 (v/v) water:acetonitrile in the original reaction vessel without
pH adjustment, followed by brief sonication and vortexing to generate turbid suspensions containing microdroplets. Similar to previous studies,
acetonitrile, a potentially prebiotic solvent, was incorporated into the solvent since a pure water solvent did not result in signiﬁcant microdroplet
formation.[11]Optical Microscopy: Following rehydration of the relevant polymerization products in 500 μL 4:1 (v/v) water:acetonitrile, 40 μL of the fresh
droplet solution was added to 40 μL of either water or a salt stock solution (of concentration twice that of the ﬁnal indicated salt concentration,i.e., 200 × 10−3 m salt stock solution was added for samples with a ﬁnal
indicated salt concentration of 100 × 10−3 m), to achieve a ﬁnal mixture of
the indicated salt concentration. Then, 3.5 μL of each mixed salt-droplet
sample was applied to a slide glass (76 × 26 × 1 mm, Matsunami Glass,
Kishiwada-shi, Osaka, Japan) into a vacated area within a double-sided
tape ring, produced by punching a hole through strong-type double-sided
tape (Naisutakku, Nichiban KK, Tokyo, Japan). This was then covered by asecond glass coverslip (No. 1 18 × 18 mm, Matsunami Glass) to prevent
evaporation. Optical microscopy images were acquired with a DM5500 B
upright epiﬂuorescence microscope (HC PL FLUOTAR 40×/0.80 PH2 air
objective, Leica, Wetzlar, Germany) with the Leica LAS X software and analyzed using FIJI (Fiji is Just ImageJ, http://ﬁji.sc).
Turbidity Measurements: Following rehydration of the relevant polymerization products in 500 μL 4:1 (v/v) water:acetonitrile, 40 μL of the
fresh droplet solution was added to 40 μL of either water or a salt stock solution to achieve a ﬁnal mixture of the indicated salt concentration. Then,
40 μL of each mixed salt-droplet sample was pipetted into a separate wellof a 384-well glass-bottom plate (Corning 4581, USA). Optical density at
600 nm was then measured using a Multimode Plate Reader (Enspire 2300
Multilabel reader, PerkinElmer, Singapore) at a controlled temperature of
25 °C, with 5 s orbital shaking at 200 rpm. The turbidity was recorded fora period of 2 h over 6 acquisitions (20 min each). Each measurement was
repeated three times.
Dynamic Light Scattering Measurements: The average diameter ofpolyester microdroplets were determined using a DelsaMax Core dynamic light scattering (DLS) instrument (Beckman Coulter, Brea, CA, USA)equipped with a 100 mW laser (660.9 nm) and 90° detector. Following rehydration of the relevant polymerization products in 500 μL 4:1 (v/v) water:acetonitrile, 40 μL of the fresh droplet solution was added to 40 μL ofeither water or a salt stock solution to achieve a ﬁnal mixture of the indicated salt concentration. Then, 45 μL of each mixed salt-droplet sample
was separately loaded into a 45 μL quartz microcuvette, and a cap was
put on top. The intensity of scattered light was recorded for a period of
2 h over six acquisitions (20 min each). Each measurement was repeatedthree times. The software package DelsaMax 1.0.0.23 was used to analyze
the data and to calculate the mean particle size.
Zeta Potential Measurement: Zeta potential was measured using a
Horiba SZ-100 Nanoparticle Analyzer (Horiba, Kyoto, Japan). Following
rehydration of the relevant polymerization products in 500 μL 4:1 (v/v) water:acetonitrile, 300 μL of the fresh droplet solution was added to 300 μL
of either water or a salt stock solution, to achieve a ﬁnal mixture of theindicated salt concentration. 300 μL of the mixture was then transferredto the appropriate sample cuvette, and analyzed on the instrument. The
test temperature was set at 25 °C. Each measurement was repeated six
times.

Small Methods 2023, 7, 2300119

Inductively Coupled Plasma Mass Spectrometry: ICP-MS analyses were
performed with He gas in kinetic energy discrimination mode using a single quadrupole (SQ-KED mode). A microﬂow PFA nebulizer (PFA-20, ESI,
USA) was used with a self-aspiration system. The sample uptake time was
set to 80 s, of which the actual integration time is 50 s. The dwell time ofeach target ion (Na+ , K+ , Mg2+ and Ca2+ ) is 0.01 s. The analytical method
contains 19 isotopes of 15 elements, including the target elements. The
calibration curve method was applied to determine ion concentrations.
As a standard solution for analysis, a mixture of the same salt solution
used for incubation was measured once for every two samples as a standard material. This standard was prepared by mixing 1 mL each of 20 ×
10−3 m salt solutions of Na+ and K+ and 200 × 10−3 m salt solutionsof Mg2+ and Ca2+ , and diluting 10 times twice with 0.5 m HNO3 , which
was prepared with distilled nitric acid (EL grade 69%, Kanto Chemicals)and USQ water (further deionized Milli-Q water), to a ﬁnal concentrationof 0.05 × 10−3 m for Na+ and K+ and 0.5 × 10−3 m for Mg2+ and Ca2+ .
The concentrations of standard solutions and samples (“initial mixture,”“supernatant,” and “droplets”) for ICP-MS analysis were adjusted so that
all ions were detected by the same detector (sample details are described
below).
For “initial mixture” samples (without separation of droplets from supernatant), poly(PA) homopolyester microdroplet solutions were ﬁrst prepared by dissolving the reaction products in Eppendorf tube in 500 μL 4:1(v/v) water:acetonitrile, followed by brief sonication and vortexing. Then,an equivalent volume of water or chloride salt solutions (NaCl, KCl, MgCl2 ,and CaCl2 ) to ﬁnal salt concentrations of 1, 10, or 100 × 10−3 m was added
to the droplet mixture, followed by vortexing. Then, 200 μL of each sample was pipetted into diﬀerent polypropylene bottles (8 mL size, Thermo
Scientiﬁc Nalgene) respectively for further sample processing. Each sample mixture was then diluted by a diﬀerent factor to match the detection/sensitivity of the instrument. For “initial mixture” sample mixturescontaining only water, 1 × 10−3 m NaCl, 1 × 10−3 m KCl, 10 × 10−3 m
MgCl2 and 10 × 10−3 m CaCl2 a 3800 μL of 0.5 m HNO3 solution wasadded into each respective sample bottle to reach a ﬁnal dilution factor of
20. For “initial mixture” sample mixtures containing 10 × 10−3 m NaCl,
10 × 10−3 m KCl, 100 × 10−3 m MgCl2 and 100 × 10−3 m CaCl2 , 7800 μL of
0.5 m HNO3 solution was added to each respective sample bottle; then,
200 μL of the diluted mixtures with HNO3 were further diluted with 800 μL
of 0.5 m HNO3 solution so that the ﬁnal dilution factor became 200. For“initial mixture” sample mixtures containing 100 × 10−3 m NaCl and 100 ×10−3 m KCl, 7800 μL of 0.5 m HNO3 solution was initially added to each
respective sample bottle; then, 200 μL of the diluted mixtures with HNO3
were further diluted with 9800 μL 0.5 m HNO3 solution, so that the ﬁnal
dilution factor became 2000. Finally, for the “initial mixture” sample mixtures containing 1 × 10−3 m MgCl2 and 1 × 10−3 m CaCl2 , 800 μL of 0.5 m
HNO3 solution was added to each respective sample bottle to reach a ﬁnal dilution factor of 5. In all cases, 1 mL of each “initial mixture” sample
solution was prepared for further analysis by ICP-MS.
Meanwhile, “supernatant” and “droplet” solutions were prepared in thefollowing way. After removing 200 μL as an “initial mixture” aliquot, theremaining sample (800 μL) was incubated overnight and centrifuged (HITACHI High-Speed Micro Centrifuge model CF16RN himac, Tokyo, Japan)
at 5000 rpm for 15 min at 23 °C; the droplets accreted to the bottom of the
Eppendorf tube. The supernatants were subsequently separated from thedroplets by pipette. Then, for “supernatant” sample mixtures with water(no salts added), 1 × 10−3 m NaCl and 1 × 10−3 m KCl, 780 μL of each supernatant solution was ﬁrst diluted with 7020 μL of 0.5 m HNO3 solution;
then, 500 μL of the diluted mixtures with HNO3 were further diluted with
500 μL of 0.5 m HNO3 solution so that the ﬁnal dilution factor became 20.
For “supernatant” sample mixtures containing 10 × 10−3 m MgCl2 and10 × 10−3 m CaCl2 , 500 μL of each supernatant solution was ﬁrst diluted
with 9500 μL of 0.5 m HNO3 solutions so that the ﬁnal dilution factor became 20. For “supernatant” sample mixtures containing 10 × 10−3 m NaCland 10 × 10−3 m KCl, 780 μL of each supernatant solution was ﬁrst diluted
with 7020 μL of 0.5 m HNO3 solution; then, 200 μL of the diluted mixtureswith HNO3 were further diluted with 3800 μL of 0.5 m HNO3 solution sothat the ﬁnal dilution factor became 200. For “supernatant” sample mixtures containing 100 × 10−3 m MgCl2 and 100 × 10−3 m CaCl2 , 500 μL of

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each supernatant solution was ﬁrst diluted with 4500 μL of 0.5 m HNO3
solution; then, 200 μL of the diluted mixtures with HNO3 were further diluted with 3800 μL of 0.5 m HNO3 solution so that the ﬁnal dilution factor
became 200. For “supernatant” sample mixtures containing 100 × 10−3 m
NaCl and 100 × 10−3 m KCl, 780 μL of each supernatant solution was ﬁrst
diluted with 7020 μL of 0.5 m HNO3 solution; then, 200 μL of the diluted
mixtures with HNO3 were further diluted with 9800 μL of 0.5 m HNO3 solution; ﬁnally, 250 μL of the twice-diluted mixtures with HNO3 were again
diluted with 750 μL of 0.5 m HNO3 solution so the ﬁnal dilution factor
became 2000. For “supernatant” sample mixtures containing 1 × 10−3 m
MgCl2 and 1 × 10−3 m CaCl2 , 500 μL of each supernatant solution was diluted with 2000 μL of 0.5 m HNO3 solution so that the ﬁnal dilution factor
became 5. In all cases, 1 mL of each “supernatant” sample solution was
prepared for further analysis by ICP-MS.
For the remaining accreted droplets after removal of supernatant, 15 μL
of each sample was ﬁrst dissolved in 100 μL acetonitrile. For “droplet”
samples containing water and 1 × 10−3 m NaCl, KCl, MgCl2 or CaCl2 ,500 μL of 0.5 m HNO3 solution was added to each acetonitrile-dissolved
sample so that the ﬁnal dilution factor became 41. For “droplet” samples containing 10 × 10−3 m NaCl, KCl, MgCl2 or CaCl2 , 900 μL of 0.5 m
HNO3 solution was ﬁrst added to each acetonitrile-dissolved sample;
then, 100 μL of the HNO3 -diluted mixtures were further diluted with 900 μL
of 0.5 m HNO3 solution so that the ﬁnal dilution factor became 677. Finally,
1 mL of each “droplet” sample solution was prepared for further analysis
by ICP-MS.
For poly(Malic-PA) and poly(4a2h-PA) heteropolyester microdroplets inthe presence of 100 × 10−3 m of each of the four chloride salt solutions,the preparation steps for ICP-MS measurements on “initial mixture” and“supernatant” samples were identical to the preparation of the poly(PA)homopolyester samples above. For the “droplet” sample solutions, 10 μL
of each sample was ﬁrst dissolved in 100 μL acetonitrile and then preparedusing the same steps as for poly(PA) homopolyester samples for further
analysis by ICP-MS.
For blank determination of monomers, 500 × 10−3 m pure PA, Malic
and 4a2h were prepared after weighing the powder and dissolution in
10 mL USQ water. Each monomer solution was diluted with 1 m HNO3
solution at a 1 to 1 volume ratio (1:1 v/v), and then subjected directly toanalysis by ICP-MS.
All “initial mixture” and “supernatant” samples were measured in triplicate (separate experiments) and all “droplet” samples were measured intriplicate, but each of the three experiments included two technical replicates (for a total of six acquisitions for each “droplet” sample); the technical replicates were ﬁrst averaged to obtain the measured cation value
for each experimental run, and the averaged values for each of the threeexperimental replicates were used for further analysis.
MicroRaman Spectroscopy: A laser Raman microspectrometer (RAMANtouch, nanophoton, Osaka, Japan) was used to measure the Raman
spectrum of one region of interest inside and one region of interest outside
one individual polyester microdroplet (roughly 10 μm in diameter) underan objective lens. 40 μL of each indicated polyester microdroplet solution(after dilution in 500 μL 4:1 (v/v) water:acetonitrile) and 40 μL of wateror respective salt solution (for a ﬁnal salt concentration of 100 × 10−3 m)were freshly mixed, followed by centrifugation (15 000 rpm for 1 min at 23°C; Model 3780; Kubota Co., Tokyo, Japan). Following the same preparation process in optical microscopy observations, 3.5 μL of the polyester
solutions were pipetted into the vacated area covered by a second glass
coverslip to prevent evaporation and then subjected to microRaman analysis. The excitation laser was a green laser with a wavelength of 532 nm.
Each Raman spectrum was obtained with 60 s exposure time and no accumulation. The excitation power was roughly 11 mW during sample measurement. Confocal experiments were carried out with a pinhole apertureof 50 μm. The measurement spot size was about 2 μm using an objective
lens (Nikon, 20X, NA 0.45). Measurements were made at three diﬀerent
locations inside and outside the same individual droplet and average values were calculated.
Statistical Analysis: 1) Preprocessing of data was performed for
MALDI-MS spectra as described above. Other data were not preprocessed.
2) All relevant data are presented as mean, with error bars indicating stan-

Small Methods 2023, 7, 2300119

dard deviation. 3) Sample size for each analysis is indicated as describedabove. 4) Statistical diﬀerences between two sets of parallel data were carried out with comparisons via a two-tailed paired t-test. Statistically signiﬁcant diﬀerences were designated as p-values <0.01. Statistically signiﬁcantdiﬀerences are indicated by *p < 0.05; **p < 0.01. 5) All statistical t-test
analyses were performed by Microsoft Excel for Mac (version 16.71). All
statistical curves were plotted under Origin 2022 (64-bit) SR1.

Supporting Information
Supporting Information is available from the Wiley Online Library or fromthe author.

Acknowledgements
Experiments at the Okayama University Institute of Planetary Materials(IPM) performed by C.C., C.S., T.K., K.K., E.N., C.P. and T.Z.J. were supported by a joint research program carried out at the Institute for Planetary Materials, Okayama University, supported by “Joint Usage/ResearchCenter” program by MEXT, Japan, to C.C., R.A., R.Y., T.Z.J., T.K., E.N., K.K.,
and C.P. R.Y., is further supported by Japan Society for the Promotion of
Science (JSPS) Grant-in-aid 21K14029. The work of A.A. is supported by
the Science and Technology Development Fund, Macau SAR, China (Fileno. SKL-LPS(MUST)-2021-2023) and by the Macau University of Scienceand Technology Faculty Research Grant (Project Grant No. FRG-22-079LPS). A.W. is supported by an Australian Research Council Discovery Early
Career Award DE210100291. K.C. is supported by the Research Encouragement Grant (GGPM-2021-057) by the National University of Malaysia andVisitor Grant (ZF-2022-008). J.H. is supported by Pioneer Hundred Talents Program of Chinese Academy of Sciences and CIFAR Azrieli Global
Scholarship. T.Z.J. is further supported by JSPS Grants-in-aid 18K14354
and 21K14746, a Tokyo Institute of Technology Yoshinori Ohsumi Fundfor Fundamental Research, a Start-up Grant from the Earth-Life Science
Institute (ELSI), the Mizuho Foundation for the Promotion of Sciences
and by the Assistant Staﬃng Program by the Gender Equality Section, Diversity Promotion Oﬃce, Tokyo Institute of Technology. C.C., R.Y., R.A.,
and T.Z.J. are members of ELSI of the Tokyo Institute of Technology, whichis sponsored by a grant from the Japan Ministry of Education, Culture,
Sports, Science and Technology as part of the World Premier International
Research Center Initiative. The authors thank M. Koizumi (The Titech Materials Analysis Division) for assistance with MALDI, Y. Nakazono (Division of Platform for Analytical Instrument at Titech) for assistance withsurface potential measurement, and K. Tanaka (PML Lab, IPM, Okayama
University at Misasa) for assistance with ICP-MS experiments.

Conﬂict of Interest
The authors declare no conﬂict of interest.

Author Contributions
C.C. and T.Z.J. conceived the research. C.C. and T.Z.J. designed the experiments with helpful discussions with all other authors. C.C. and R.A.
performed the polyester synthesis and optical image observations; C.C.
performed MALDI-TOF-MS analysis under the guidance of K.C. and T.Z.J.;
C.C. performed OD600, DLS and zeta-potential measurement experiments and data analysis; C.C. and M.I. performed the MicroRaman experiment and analysis; C.C. and C.S. performed the ICP-MS experiments
and data analysis with the assistance of R.Y. and T.Z.J. in the clean roomat the pheasant memorial laboratory (PML), Institute for Planetary Materials (IPM), Okayama University at Misasa; C.C. wrote the original draft;
C.C. and T.Z.J. reviewed and edited the subsequent drafts and the ﬁnal
manuscript. All authors have discussed the results and commented on
the paper.

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www.small-methods.com

Data Availability Statement
The data that support the ﬁndings of this study are available in the supplementary material of this article.

Keywords
membraneless compartments, origins of life, phase separation, prebiotic
chemistry, protocells
Received: January 29, 2023
Revised: April 23, 2023
Published online: May 18, 2023

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