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The development of a high-sensitivity calorimeter relies on abuse of the thermal conductance and minimum credible temperature. Antecedent two-dimensional film designs still accept ample thermal conductance. In our design, we activated a credible (1D) abeyant microfluidic approach with a baby cross-sectional breadth and ample breadth (2L = 5.0 mm) to accomplish low advice and radiation thermal conductance (Fig. 1a). The microfluidic approach is composed of parylene, which has aerial automated strength, acceptable beheld transparency, and, best importantly, low thermal appliance (Supplementary Fig. 2b) compared with added abstracts broadly acclimated for dent calorimeters (e.g., Si3N422,24,26, glass25, Si27). The parylene courage band had a amplitude of 120 μm and array of 12 μm and formed the microfluidic tube (Fig. 1b). The microfluidic approach had a cross-sectional breadth of 35 × 50 μm2, which is accordant with best corpuscle sizes (<35 μm in diameter). The absolute tube anatomy was abeyant on a silicon dent with a ~ 200-μm gap. Lastly, the absolute accessory was placed in a high-vacuum ambiance (<0.1 mT)16 to annihilate alteration calefaction accident (Fig. 1d, e). The fluids into and out of the abounding approach were alien via exhaustion feedthroughs. Photographs of the bureaucracy are credible in Supplementary Fig. 6.

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a SEM angel of calorimeter with a 5-mm continued (2L) credible abeyant microfluidic channel. The calorimeter is amidst by a exhaustion ambiance to abate calefaction loss. Added than 10 samples (n > 10) with the above geometry were reproducibly fabricated. b Cross-sectional schematic analogy of calorimeter. c Photograph of boilerplate of abeyant channel, including barometer junctions of the Bi–Pt thermopile and a Pt heater. d Schematic analogy of thermal insulation architecture of altitude belvedere with three temperature-controlled layers (outer chamber, exhaustion chamber, and sample stage). e Schematic analogy of apparatus of the exhaustion chamber. Photographs of the bureaucracy are credible in Supplementary Fig. 6.

A Bi–Pt thermopile and Pt boiler were microfabricated and dent in the parylene band beneath the microfluidic approach (Fig. 1c). The thermopile abstinent the temperature aberration (ΔT) amid the barometer junctions (Tmea) in the boilerplate of the approach and the advertence junctions (Tref) on the Si dent (Fig. 1a). The Pt boiler was acclimated to characterize the thermal conductance of the abounding approach by applying joule heating to the channel. The thermopile was calibrated by adopting the temperature of the alliance to accepted temperatures with the Pt boiler (Fig. 1c) and barometer the voltage of the thermopile. The thermopile was advised by because the tradeoff amid the thermal conductance and temperature sensitivity: accretion the cardinal of thermopile junctions leads to bigger temperature apprehension acuteness but additionally increases the thermal conductance. Likewise, abbreviation the attrition of the thermopile decreases the electrical babble (e.g., Johnson or 1/f noise) but after-effects in college thermal conductance. Our optimized thermopile was composed of four pairs of Bi and Pt attenuate films (Fig. 1c and Supplementary Fig. 1b), and the root-mean-square (rms) voltage babble of the altitude arrangement was 19 nV, which includes noises from the thermopile, an operational amplifier (CS3002, Cirrus Logic), and a low-pass clarify (cutoff frequency: 0.016 Hz), as credible in Supplementary Fig. 4a.

The all-embracing thermal conductance of our calorimeter including the above apparatus and baptize in the microfluidic approach was estimated as a action of the bisected approach breadth (L) appliance a fin blueprint (see Supplementary Note 1 for derivation):

$$G = 2mkA_cleft( {frac{{e^{mL} e^{ – mL}}}{{e^{mL} – e^{ – mL}}}} right)$$


where the fin connected (m = sqrt {frac{{hP}}{{kA_{mathrm{c}}}}}), k is the thermal conductivity, Ac is the cross-sectional breadth of the microfluidic channel, P is the alien ambit of the microfluidic channel, and h is the radiation heat-transfer accessory (h = 4εσTavg3, breadth σ is the Stefan–Boltzmann constant, ε is the emissivity, and Tavg is the boilerplate temperature). Blueprint (1) shows that the all-embracing thermal conductance is saturated at L = 2.5 mm, breadth radiation calefaction accident becomes added important than the advice calefaction accident because of the ample credible breadth (Supplementary Fig. 3a). The estimated all-embracing thermal conductance with L of 2.5 mm is accepted to be 2.48 μW K−1, including the courage parylene layers, baptize central the microfluidic channel, and Bi–Pt thermopile. It is account acquainted that ~48% of the absolute thermal conductance comes from the baptize approach (Supplementary Fig. 3b), which is bare for the connected comestible and oxygen supply. We additionally optimized the geometry to ensure the temperature accord about the corpuscle (Supplementary Fig. 3c) and automated candor of the channel–substrate alliance (Supplementary Fig. 3d).

The bogus accessory was loaded assimilate our altitude belvedere (Fig. 1d, e), which was advised to abbreviate the baseline temperature alluvion and accommodate a exhaustion environment. The temperature adherence is abnormally important in single-cell calorimetry because alien agitations such as ablaze beam from a microscope acclimated to anticipate the corpuscle in absolute time and aqueous breeze are inevitable. We minimized the temperature alluvion by appliance three levels of thermal insulation and temperature-control layers (Fig. 1d) as able-bodied as a abiding and hermetically closed aqueous ascendancy arrangement (Fig. 1e). By implementing these all-encompassing thermal and abounding ascendancy schemes, we were able to accomplish a baseline temperature adherence of our calorimeter of aural 80 μK for added than 10 h (Fig. 2a) beneath the action of microscope illumination. We additionally showed that the thermal conductance and temperature adherence were agnate back the aqueous in the microchannel was abounding at a acceleration of 0.1 mm s−1 (Supplementary Fig. 7a, c), which was bare to accommodate acceptable comestible and oxygen accumulation to distinct Tetrahymena beef (see abutting section).

a Temperature aberration of microfluidic approach abstinent by Pt/Bi thermopile for 10 h beneath microscope illumination. Agnate after-effects with 0.1 mm s−1 aqueous breeze are credible in Supplementary Fig. 7a, c. b Temperature acceleration of calorimeter in acknowledgment to Joule heating from anchored Pt heater. The beggarly ΔT and rms temperature babble (Tnoise) based on the temperature altitude after-effects calm every 0.5 s for 5 min (n = 600), are displayed as a action of captivated ability (Qabs). Tnoise was 80 μK and the thermal conductance of the calorimeter was 2.51 μW K−1 (G = dQabs/dΔT, extracted from the above artifice with a added ambit of Qabs credible in the inset) agnate to calorimetry acuteness of 0.2 nW (Qs = G·Tnoise). c Comparison of G (y-axis) and Qs (x-axis) of antecedent calorimeters16,17,18,19,20,21,22,23,24,25,26,27,28 and ours in this work. The numbers abutting to the abounding circles are the advertence numbers of the affidavit advertisement the agnate calorimeters. The dejected accumbent confined appearance the accepted metabolic ante of archetypal cells. Source abstracts for 2a, b are provided as a Source Abstracts file.

The acuteness of our calorimeter was approved by heating the abeyant approach with the anchored Pt boiler and ecology the voltage from the Bi–Pt thermopile. The heating ability captivated by the barometer junctions (Qabs) gradually added from 0 to 4 μW, and the voltage achievement from the thermopile was calm for 5 min at anniversary captivated power. The voltage achievement from the thermopile was adapted to a temperature arresting (ΔT) appliance the Seebeck accessory (S = 229 μV K−1), and the boilerplate ΔT and rms temperature babble (Tnoise) of anniversary altitude are advised in Fig. 2b. The abstinent thermal conductance of our calorimeter was 2.51 μW K−1 (G = dQabs/dΔT), which is connected with the analytic admiration acquired appliance Eq. (1) (2.48 μW K−1). This thermal conductance is about six times lower than that of the advanced dent calorimeter (16 μW K−1)16. Tnoise was 80 μK for 5 min, and the agnate acuteness of our single-cell calorimeter was 0.2 nW (Qs = G × Tnoise). This acuteness is about one adjustment of consequence bigger than that of advanced on-chip calorimeters (4.2–13 nW)16,26, as abbreviated in Fig. 2c. The accepted metabolic ante of several beef additionally credible in Fig. 2c; it is credible that our calorimetry belvedere accomplished acceptable acuteness to admeasurement the metabolic aggregate of distinct beef such as protozoa29,30,31 or angry beastly cells32, which accept not been able to be abstinent by antecedent calorimeters16,17,18,19,20,21,22,23,24,25,26,27,28.

As a affirmation of our calorimeter and altitude platform, we abstinent metabolic calefaction bearing from distinct Tetrahymena thermophila SB210. Tetrahymena is broadly acclimated as a archetypal corpuscle band to abstraction beastly beef because it has aerial affinity with beastly beef not alone responding to beastly hormones but additionally synthesizing agnate molecules (e.g., insulin)33. Therefore, their metabolism has been advised by ecology their oxygen consumption34,35,36,37 or metabolic heat29,30,31 or the admeasurement of their mitochondria;38 however, none of the absolute altitude platforms accept enabled single-cell metabolic aggregate analysis. Appliance our high-sensitivity single-cell calorimeter, we were able to admeasurement the metabolic aggregate of distinct Tetrahymena with sub-nW resolution.

A key claiming to admeasurement Tetrahymena (and added cells) is the charge to allurement alone beef in the microfluidic approach and accumulation acceptable nutrients and oxygen to the trapped cells. Notably, Tetrahymena are difficult to allurement because they move to acquisition nutrients appliance cilia. To allurement Tetrahymena, we activated the alluring corpuscle accoutrement arrangement schematically illustrated in Fig. 3a (see “Methods”). As ahead shown39, Tetrahymena can booty up adamant oxide nanoparticles, and their movement can afresh be controlled by an alien alluring field. The attendance of adamant oxide nanoparticles was acutely arresting in the cytoplasm in the Tetrahymena (Fig. 3c), admitting the Tetrahymena afterwards the particles displayed accustomed cytoplasm (Fig. 3b). It is account acquainted that adamant oxide nanoparticles are not accepted to blemish any cellular activity40. With the appliance of an alien alluring field, the ingested adamant oxide nanoparticles produced an induced alluring dipole and were accumbent with the administration of the alluring force, as credible in Fig. 3d. The Tetrahymena with the particles can afresh be admiring and eventually trapped in the attendance of the alien alluring field. By adjustment the boilerplate of the calorimeter with the interface of the two magnets (Figs. 1e and 3a), we were able to abode and allurement alone Tetrahymena beef sitting aloft of the thermopile alliance in the boilerplate of the microfluidic channel.

a Analogy of alluring corpuscle trapping. The alien alluring acreage generated by two magnets trapped a distinct cell, which is internalized with iron oxide nanoparticles at the interface of the magnets, breadth the accomplished alluring acreage occurred. b Tetrahymena afore internalizing adamant oxide nanoparticles. c Tetrahymena afterwards internalizing adamant oxide nanoparticles. d Tetrahymena with the adamant oxide nanoparticles beneath alien alluring field. Added than bristles (n > 5) agnate images of beef with nanoparticles were taken. e Action of single-cell metabolic aggregate measurement: (i) bang of a distinct Tetrahymena through the microfluidic channel, (ii) accoutrement of the corpuscle in the boilerplate of the abeyant tube by the alluring acreage and altitude of the temperature with the corpuscle (Tcell), (iii) FCCP bang through the microfluidic approach to activate corpuscle metabolism and altitude of the temperature with the dispatch (TFCCP), and (iv) corpuscle abatement with a beating breeze to admeasurement baseline temperature (Tbase). f Adumbrative aftereffect of the temperature arresting during the single-cell measurement. Solid band represents the raw abstracts of the temperature signal. Dashed curve represent the boilerplate ethics afore and afterwards corpuscle abatement (Tcell and Tbase, respectively) The inset photograph shows the Tetrahymena trapped by the alluring field during the measurement. Source abstracts for 3f are provided as a Source Abstracts file.

As a distinct Tetrahymena was trapped, nutrients and oxygen had to be supplied to advance their accustomed metabolism. Notably, afterwards a connected accumulation of oxygen, the aggregate of oxygen absolute in the microfluidic approach and transported to Tetrahymena would accept been bound because of the baby aggregate and cross-sectional area. The oxygen burning of Tetrahymena is accepted to be 0.1–1.0 nLO2 cell−1 h−134; however, our assay adumbrated that the aggregate of oxygen in the approach was not acceptable for a distinct Tetrahymena (Supplementary Fig. 5). Furthermore, antecedent studies accept credible that fatigued protozoa accept a abate metabolic aggregate than accustomed protozoa29,34. To accumulation acceptable nutrients and oxygen, we activated a 0.1 mm s−1 breeze of the advance boilerplate to the trapped Tetrahymena. The oxygen accumulation by the breeze was estimated to be ~3.4 nL O2 h−1 at this breeze rate, which is a few times beyond than the estimated oxygen burning of distinct Tetrahymena. This low breeze aggregate was bereft to affected the alluring force accoutrement Tetrahymena; therefore, the trapped Tetrahymena were not confused by the breeze during the altitude (Supplementary Movie 1 (cell trapping)). In addition, the breeze did not accept a above aftereffect on the calorimeter sensitivity. We bent the thermal conductance of the calorimeter with the breeze appliance the above adjustment declared in Fig. 2b to verify the aggregate of accessible advection calefaction alteration by the flow. The aftereffect adumbrated that the advection calefaction alteration was about negligible; thermal conductance with the breeze was alone added by ~4% compared with the no-flow action (from 2.51 to 2.62 μW K−1, Supplementary Fig. 7a). Furthermore, this low-speed connected breeze was credible to accept negligible aftereffect on the temperature adherence in the abounding channel (Supplementary Fig. 7c). This was mainly accomplished by accumulation a 2 cm-long convolute microfluidic approach on the temperature-controlled Si substrate to preheat the aqueous and balance its temperature afore it enters the abeyant approach (Supplementary Fig. 1c). The temperature arresting from the abeyant calorimeter alcove afore and afterwards injecting the aqueous (at connected acceleration of 0.1 mm s−1, agnate to 0.175 nL s−1 breeze rate) was the same, apprehend for the baby acting bead for a continuance of about 1 min afterwards the bang (Supplementary Fig. 4e). In contrast, pulsed breeze with a aerial breeze aggregate can account acting temperature reduction. For example, pulsed aqueous bang with ~ 10 nL s−1 breeze aggregate acquired ~ 0.8 mK acting temperature bead (Supplementary Fig. 4c), because the convolute approach is not continued abundant to acquiesce a acceptable abode time for high-rate pulsed breeze to ability thermal calm (about 3.5 s compared with 200 s for 0.175 nL s−1 flow). We empiric this burning temperature bead due to the pulsed aqueous bang back we removed the beef from the calorimeter for baseline altitude (Fig. 3f). This temperature bead was recovered in <1 min as anon as we adequate the breeze aggregate to the baseline akin of 0.1 mm s−1.

The action and a adumbrative aftereffect of the metabolic aggregate altitude of distinct beef are displayed in Fig. 3e, f, respectively. First, a distinct Tetrahymena was injected through the microfluidic approach and trapped on the barometer junctions by a alluring field. Then, the thermopile recorded the temperature arresting of the boilerplate of the microfluidic approach with the trapped corpuscle (Tcell) while 0.1 mm s−1 breeze of advance boilerplate supplied a acceptable aggregate of nutrients and oxygen. Afterwards the measurement, the corpuscle was removed from the calorimeter by a pulsed flow, which was abundantly able to affected the alluring force. Then, the temperature arresting afterwards the corpuscle (Tbase) was recorded as a baseline. In Fig. 3f, the abrupt temperature change at 5 min was acquired by the pulsed breeze for corpuscle removal. This altitude action is credible in Supplementary Movie 2 (cell injection) and Movie 3 (cell removal).

We auspiciously abstinent the metabolic aggregate of distinct cells. The temperature arresting abstinent from the thermopile back a corpuscle was trapped in the calorimeter (Tcell) was college than the baseline temperature afterwards removing the corpuscle (Tbase). This temperature acceleration (Tcell  − Tbase) indicates the calefaction assembly by cellular metabolic action of a distinct Tetrahymena and can be adapted into metabolic aggregate (Qcell = G × (Tcell − Tbase)). As displayed in the inset blueprint of Fig. 4a, the boilerplate basal metabolic aggregate of Tetrahymena was 2.0 ± 0.7 nW cell−1. This aftereffect avalanche aural the ambit of appear ethics (from 1 to 6 nW cell−129,30,31); however, our abstracts accommodate a abundant added absolute amount at the alone corpuscle level. As a ascendancy experiment, we additionally abstinent the calefaction bearing aggregate from asleep beef as able-bodied as abounding baptize afterwards cells, which adumbrated no temperature acceleration from the baseline (Fig. 4a). In addition, the temperature acceleration and calefaction bearing of two and three beef were additionally measured, which acutely showed an access of the calefaction bearing from ~2 nW for one corpuscle to ~4 nW for two beef and assuredly to ~ 6 nW for three cells. These abstracts accepted that the detected temperature acceleration originated from the corpuscle metabolic calefaction production. With these measurements, we added quantified the corpuscle metabolic calefaction aggregate as a action of alone corpuscle admeasurement (Fig. 4b); the after-effects are abbreviated in Fig. 4c (with all the raw abstracts for anniversary altitude presented in Supplementary Fig. 8).

a Basal metabolic aggregate (Qcell) of one (red line), two (purple line), three (blue line), and asleep beef (green line) as able-bodied as the calefaction arresting from baptize bang afterwards beef (black line). A bright access of the calefaction arresting was empiric with accretion cardinal of cells, admitting no calefaction arresting was detected for the asleep corpuscle or baptize bang afterwards cells. The inset blueprint compares the Tetrahymena metabolic aggregate from the abstract (3.1 ± 1.4 nW, breadth the ±error represents accepted deviation)29,30,31 and this assignment (2.0 ± 0.7 nW)), breadth n = 15 absolute distinct corpuscle abstracts were performed. b Single-cell metabolic aggregate for assorted sizes of Tetrahymena. The ample corpuscle (35 × 103 μm3, dejected line), boilerplate corpuscle (16 × 103 μm3, amethyst line), and baby corpuscle (9 × 103 μm3, red line) apparent metabolic ante of 3.3, 2.1, and 0.9 nW, respectively. The calibration confined in the images all represent 30 μm. n = 15 absolute distinct corpuscle images were taken, agnate to the 15 calorimetry of distinct cells. c Summary of single-cell metabolic aggregate as a action of corpuscle admeasurement (V). Atramentous dots represent alone beggarly amount of Qcell calm for 5 min from n = 15 absolute distinct corpuscle abstracts with altered cells. The x-axis absurdity confined represent accepted aberration of calefaction signals abstinent every 0.5 s for 5 min (n = 600) while the y-axis absurdity confined are based on accessible accepted aberration errors of corpuscle admeasurement altitude (see “Methods” breadth for details). The inset presents the after-effects on the logarithmic scale. The dotted band in the inset represents a three-quarter ability allometric ascent accord (Qcell ∝ V 3/4). d Adumbrative metabolic calefaction altitude aftereffect with FCCP stimulation. The corpuscle metabolic aggregate was added aloft dosing FCCP to the corpuscle at 3 min amid n = 3 absolute abstracts with altered cells. The inset shows the SRC (ratio of best metabolic rate, Qmax, to Qcell) abstinent by our single-cell calorimeter and a accepted oxygen electrode chamber on a aggregate aggregate of Tetrahymena. Source abstracts for 4a–d are provided as a Source Abstracts file.

We added approved dispatch of the metabolic aggregate by injecting the uncoupling abettor FCCP. The altitude action was the above as that credible in Fig. 3e, with the added footfall of injecting FCCP at a connected breeze aggregate of 0.1 mm s−1 into the microfluidic approach afterwards the antecedent temperature of the microchannel with corpuscle was stabilized. Afterwards accoutrement a distinct Tetrahymena, the calefaction arresting from the corpuscle adumbrated a archetypal basal metabolic rate. Back FCCP was injected into the calorimeter, the calefaction bearing from the corpuscle gradually added and eventually accomplished a best (Qmax) in 1–2 min (Fig. 4d). This angry calefaction arresting was calm for 3 min, and afresh the baseline was abstinent afresh afterwards removing the corpuscle with the pulsed flow. The basal metabolic calefaction bearing of Tetrahymena added by a agency of 2.1 ± 0.3 with FCCP stimulation. We additionally abstinent the change in the oxygen burning aggregate of a aggregate aggregate of Tetrahymena aloft FCCP dispatch appliance a Hansatech Oxytherm apparatus41. The basal and angry oxygen burning aggregate of Tetrahymena were abstinent to be 0.20 ± 0.01 and 0.39 ± 0.04 nLO2 h−1, respectively. These abstracts adumbrated that the oxygen burning aggregate added by a agency of 1.9 ± 0.1 with the FCCP dispatch (inset of Fig. 4d), which is connected with our calorimeter results.

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