With the selected light sources and buffer conditions, PSMM1–3 were tested as described in the Experimental Section. All three nanomotors displayed different fluorescence recovery after visible irradiation followed by UV irradiation ( and Table S1 in Supporting Information
). As expected, more Azo- incorporations, such as those incorporated in PSMM3, resulted in higher fluorescence recovery, indicating that increasing of the amount of Azo- moiety can introduce a higher impact from azobenzene isomerization to hairpin structure stability. Excess amount of cDNA by 5-fold was added at the end for each type of nanomotor after photoregulation in order to compare the fluorescence intensity of all PSMMs at fully open state (induced by cDNA). By setting the fluorescence intensity when azobenzene takes the transform as a baseline (0%) and the intensity after addition of excess amount of cDNA as 100%, we could estimate the number of PSMMs in the open state for each photoregulation process. In our case, we set the fluorescence intensity on close state at 488 nm as the baseline (blue curves) and the fully open state (green curves) as 100%. We were then able to set up a fluorescence recovery parameter, recovery (%), based on the three states as defined below in order to evaluate the close–open conversion efficiency
Figure 1 Fluorescence spectra of PSMM1–3 (λex = 488 nm) from top to bottom after irradiation under a 6 W UV lamp (350 nm) and a 60 W desktop lamp with 450 nm filter at 25 °C. All other conditions are the same. The blue curve is pure DNA (more ...)
IUV is the fluorescence intensity of DNA solution after UV irradiation, I0 is the fluorescence intensity after visible irradiation, It is the fluorescence intensity of DNA solution after adding extra cDNA. The higher the recovery value, the higher the amount of molecules that will be found in open state driven by photonic energy, and the better the efficiency of the conversion from close to open state. Since the reversible open to close conversion step is very fast for each of our PSMMs, we focus the recovery percentage on close to open conversion and discuss the motor efficiency accordingly.
We used the recovery (%) to compare the efficiency of energy conversion from photonic energy to molecular motion for PSMM1–3 under the same conditions. We found approximately 14.2% of recovery from trans- to cis- for PSMM1, 26.3% for PSMM2, and 54.7% for PSMM3. This result supported our assumption that multiple azobenzene incorporation will introduce higher impact to hairpin structure stability or photoregulation capability. The improvement of this recovery (%) is, however, not proportional to the number of Azo- moiety, although it does gradually increase as the number of Azo- increases. Specifically, the triple Azonanomotor (PSMM3) displayed a much higher open-to-close ratio than PSMM1 and PSMM2, which supports this argument. Furthermore, the result is consistent with a previous study of the relationship between Azo- moiety and duplex association/dissociation conversion. Because the energy barrier of azobenzene isomerization from trans- to cis- is higher than cis- to trans-, the trans- to cis- conversion requires a long UV light irradiation time to drive this conversion. Our results demonstrate that recovery (%) can reach to about 60% (±3.0%) after 20 min of UV irradiation before photobleaching of FAM fluorophore appears to become a serious problem. Thus, for realistic usage, which balances input and output energy, a UV irradiation time from 2 to 10 min is satisfactory for the PSMM running in numerous cycles without losing apparent functionality and efficiency.
Theoretical calculation of the extension and contraction forces is based on free energy and extending capability of single- and double-stranded DNA. We estimated the force based on Gibbs free energy and the distance variation from close and open structures. The L1
are approximately 10.2 and 2.2 nm and give the estimated values of two forces of 1.5 and 3.1 pN (the average forces based on our irradiation timeline), respectively (see Supporting Information g
). Noticeably, the single-stranded DNA has the nature of forming random coil in solution instead of an extended structure. Here, we estimated the sizes by free energy and persistent length when applied. The actual size variation will determine the potential motor strength. The total input photon energy on extending the nanomotor can be calculated by UV lamp power (0.197 mW) and irradiation time (5 min) with Einput
= Ps = 5.91 × 10−2
J. On the basis of our previously calculated extension force (1.5 pN) and distance (8 nm), each nanomotor has the extension work of 1.2 × 10−20
= Fs). We can regard the extension work as the output mechanical energy. Therefore, the total output work Woutput
for each type of nanomotor under our conditions can be calculated by Woutput
= [(extended molecule)%] × [total molecule number (100 nM × 120 μ
L × NA
= 7.22 × 1012
] × [woutput
]: PSMM1 (14.2%), 1.23 × 10−8
J; PSMM2 (26.3%), 2.28 × 10−8
J; and PSMM3(54.7%), 4.74 × 10−8
J. The energy conversion efficiencies (Einput
) are 2.09 × 10−7
, 3.85 × 10−7
, and 8.02 × 10−7
shows the reversibility of the PSMM3 nanomotor for ten rounds of close-open cycles. For each cycle, 1 min visible irradiation and 3 min of UV irradiation were applied. Compared to an approximate 40% decrease in cycling recovery for the previous DNA-fueled nanomachines under more favorable conditions (higher temperatures, more Azo- ratio), the cycling of PSMM3 maintains its recovery consistency and has no tendency to decrease after ten cycles. Additionally, all the cycles were performed at room temperature (25 °C) where the close state is more favored. As such, we can predict that conversion efficiency of our motor can be further improved at higher temperatures (close to Tm) as the stem is close to the transition point from duplex to single-strand state. This result demonstrates that the DNA hairpin nanomotor possesses high close-open conversion efficiency and molecular stability under current operating conditions. Repeated nanomotor cycles displayed no obvious decomposition of the motor (up to 20 cycles; data not shown). Overall, these results demonstrate a long-lasting molecular motor with high conversion efficiency using a clean energy input.
Figure 2 Cycling of close-open from Vis/UV irradiations at 25 °C by repeated visible and UV irradiations. Vis (450 nm), 1 min; UV (350 nm), 3 min. Fluorescence intensities at the maximum emission (525 nm) were recorded immediately after each irradiation. (more ...)
A comparably high fluorescence recovery seems very interesting from the perspective of energy application since it is related to energy conversion efficiency. That is, under the same operating conditions for similar molecular nanomotors labeled with F/Q, a higher fluorescence variation illustrates that higher ratios of molecules are driven from one state to another state. This comparison is applicable to hairpin structures as well as linear DNA strands as long as we normalize the conditions and set up correlated parameters. In this case, we can use the previously defined recovery percentage as the indicator of energy conversion efficiency. Under these conditions, a higher recovery percentage means that comparable DNA motor systems can convert more absorbed energy to drive the structure changes. This efficiency can therefore be related to the capability of fulfilling motorlike function. Although it is possible that different molecular structures might absorb different amounts of photon energy under the same conditions, we can still count it as part of the overall energy conversion capability for specific molecules.
In order to compare this conversion efficiency between our hairpin single-molecule nanomotors and previously investigated models, we designed a series linear DNAs, as well as another hairpin structure, using melting temperature (Tm
) as the correlated parameter for comparison. The details of the rationale underlying this assumption are discussed in Supporting Information
. The definition of Tm
for short DNA duplex can be expressed by
° and ΔS
° are the melting parameters; R
is the ideal gas constant; CDNA
is the molar concentration. For DNA sequences with the same concentrations, CDNA
is the same, while ΔS
° varies slightly. Therefore, Tm
is approximately proportional to ΔH
°, which is the energy absorbed by the DNA molecule to dissociate duplex structures. Therefore, Tm
value can be regarded as the capability of absorbing sufficient energy to dissociate DNA duplex structures. For DNA nanomotors involved in duplex dissociation, we can always use Tm
as the standard by which to evaluate the structure conversion or motor operation capability by absorbing external energy. For different duplex structures, such as hairpin and linear duplex with the same Tm
value, they are expected to display the same recovery percentage under the same photoregulation conditions.